Gnomoniopsis chinensis (Gnomoniaceae, Diaporthales), a new fungus causing canker of Chinese chestnut in Hebei Province, China

Abstract Chinese chestnut (Castanea mollissima) is an important crop tree species in China. However, branch canker and fruit rot are two kinds of severe diseases, which weaken the host and decrease chestnut production. During our investigations into chestnut diseases in China, several fungi have been confirmed as casual agents in previous studies, namely Aurantiosacculus castaneae, Cryphonectria neoparasitica, Cry. parasitica, Endothia chinensis and Gnomoniopsis daii. In this study, a new canker pathogen is introduced based on morphology, phylogeny and pathogenicity. Typical Gnomoniopsis canker sign of wide, orange tendrils emerging from hosts’ glaucous lenticels were obvious on the diseased trees in the field. Symptomatic branches or bark on stems from different chestnut plantations were sampled and isolated, then strains were identified by comparisons of DNA sequence data for the nuclear ribosomal internal transcribed spacer (ITS), partial translation elongation factor-1α (tef1) and β-tubulin (tub2) gene regions as well as morphological features. As a result, these strains appeared different from any known Gnomoniopsis species. Hence, we propose a novel species named Gnomoniopsis chinensis. Pathogenicity was further tested using the ex-type strain (CFCC 52286) and another strain (CFCC 52288) on both detached branches and 3-year-old chestnut seedlings. The inoculation results showed that Gnomoniopsis chinensis is mildly pathogenic to Chinese chestnut. However, further studies are required to confirm its pathogenicity to the other cultivated Castanea species in America, Europe and Japan.

Gnomoniopsis smithogilvyi is an important nut rot agent on chestnut nuts, an endophyte in asymptomatic flowers, leaves and stems, and a saprobe on dead burrs and branches (Crous et al. 2012;Visentin et al. 2012). Moreover, this species has been reported as a severe bark pathogen on Castanea in several countries Rai 2013, 2015;Pasche et al. 2016;Lewis et al. 2017;Trapiello et al. 2018;Lione et al. 2019). In China, Gnomoniopsis from rotten Chinese chestnut has proved to be a different species, namely Gnomoniopsis daii (Jiang and Tian 2019). In this study, we focused on the symptom, taxonomy and pathogenicity aspects of Gnomoniopsis species from cankered tissues on Chinese chestnut trees.

Sample collection and isolation
During 2016 to 2019, investigations were conducted in chestnut plantations of nine provinces/municipalities in China, including Beijing, Fujian, Hebei, Hubei, Hunan, Liaoning, Shandong, Shaanxi and Tianjin. Typical Gnomoniopsis canker symptoms were only observed in Hebei Province (Fig. 1). Symptomatic barks from stems and cankered branches were collected in brown paper bags and transported to the laboratory for fungal isolations and further study. Single conidial isolates were acquired from asexual fruiting structures by removing a mucoid conidial mass from pycnidial ostioles, and spreading the suspension on the surface of potato dextrose agar (PDA; 200 g potatoes, 20 g dextrose, 20 g agar per L). Agar plates were incubated at 25 °C to induce germination of conidia. After inoculation for up to 36 h, single germinating conidia were then transferred to clean plates under a dissecting stereomicroscope with a sterile needle. Specimens and cultures were deposited and maintained in the Museum of Beijing Forestry University (BJFC) and China Forestry Culture Collection Center (CFCC), Beijing, China, respectively.

DNA extraction and phylogenetic analysis
Genomic DNA was extracted from mycelium grown on PDA using a CTAB (cetyltrimethylammonium bromide) method (Doyle and Doyle 1990). Three partial loci, including the 5.8S nuclear ribosomal DNA gene with the two flanking internally transcribed spacer (ITS) regions, the translation elongation factor 1a (tef1), and the β-tubulin gene 2 (tub2), were amplified using the following primer pairs: ITS1 and ITS4 for ITS (White et al. 1990), EF1-728F and EF1-1567R for tef1 (Carbone and Kohn 1999), and Bt2a and Bt2b for tub2 (Glass and Donaldson 1995). The PCR conditions were: initial denaturation step of 5 min at 94 °C, followed by 35 cycles of 30 s at 94 °C, 50 s at 48 °C (ITS) or 54 °C (tef1) or 52 °C (tub2), and 1 min at 72 °C, and a final elongation step of 7 min at 72 °C. The PCR amplification products were scored visually by electrophoresis in 2 % agarose gels. The DNA sequencing was performed using an ABI PRISM 3730XL DNA Analyzer with BigDye Terminater Kit v.3.1 (Invitrogen) at the Shanghai Invitrogen Biological Technology Company Limited (Beijing, China). To assess the phylogenetic position of our isolates within the genus Gnomoniopsis, phylogenetic analyses were performed based on combined ITS, tef1 and tub2 sequence data, with Sirococcus castaneae (CBS 142041) and Apiognomonia errabunda (CBS 342.86) selected as outgroup taxa. The GenBank accession numbers of sequences used in the analysis are given in Table 1, which were aligned and edited manually in MEGA6 (Tamura et al. 2013). Maximum likelihood (ML) analysis was used for phylogenetic inferences of the concatenated alignments. ML analysis was implemented on the CIPRES Science Gateway portal using RAxML-HPC BlackBox v. 8.2.10 (Stamatakis 2014).

Morphological identification and characterization
Species identification was based on morphological features of the asexual fruiting bodies produced on infected plant tissues, supplemented by cultural characteristics. Hence, cross-sections were prepared by hand using a double-edge blade. Morphological characteristics of the fruiting bodies including: size of conidiomata and locules; size and shape of conidiophores and conidia were determined under a Nikon AZ100 dissecting stereomicroscope. More than 20 fruiting bodies were sectioned, and 50 conidia were selected randomly for measurement using a Leica compound microscope (LM, DM 2500). Cultural characteristics of isolates incubated on PDA in the dark at 25 °C were recorded, including the colony color and pycnidium structures (Rayner 1970).

Pathogenicity trials
Two isolates of Gnomoniopsis chinensis (ex-type strain: CFCC 52286; CFCC 52288) were used for inoculations, and agar plugs were used as the negative control. Isolates were grown on PDA for five days at 25 °C before the tests. Inoculations were performed on detached branches and 3-year-old seedlings of Castanea mollissima, respectively. The detached branches and young seedling were collected from Hebei Province where the disease is emerging. The healthy chestnut branches (2 cm in diameter) were sampled from an adult chestnut tree and cut into pieces of 20 cm length. A total of 30 fresh and healthy branches and 15 seedlings were used for the pathogenicity tests. Ten branches and five seedlings were inoculated with each isolate and the negative control. For incubations, incisions were made on the middle of the detached branches and 1 cm above the midpoint of the seedling stem to expose the cambium using a 5-mmdiameter cork borer. Discs of agar were cut from the actively growing margins of the cultures and these were placed into wounds of the same size on the chestnut barks. Inoculated wounds and ends of inoculated branches were sealed with parafilm to reduce desiccation and the chance of contamination. The tested seedlings and branch segments were maintained in the greenhouse randomly at 25 °C under natural light conditions. Detached branches were inoculated in November 2017, and the young seedlings were tested in July 2019. The results from detached branches were evaluated after one month, and seedlings after three months, by measuring the lengths of the lesions on the cambium. The re-isolations were made from the resultant lesions from all tested branches and seedlings by cutting small pieces of discolored xylem and placing them onto the PDA plates. Re-isolations were identified based on morphology on PDA and ITS sequences. Differences among isolates in lesion length were analyzed by one-way analysis of variance (ANOVA) followed by least significant difference (LSD) tests. Statistical analysis was carried out by R software (version 3.4.3.) and considered as significant at p < 0.05.

Pathogenicity trials
One month after inoculation on detached branches, the two Gnomoniopsis chinensis isolates produced lesions in the cambium of detached chestnut branches. In contrast, there was no lesion development in any of the negative control inoculations (Fig. 5). The lesion size of the two Gnomoniopsis chinensis isolates (CFCC 52286 and CFCC 52288) showed no significantly difference, while both of them were significantly longer than the negative control (P < 0.05 ). Gnomoniopsis chinensis was consistently re-isolated from lesions.
Three months after inoculation on young seedlings, two isolates Gnomoniopsis chinensis and the negative control, produced minor lesions (Fig. 6). Statistical analyses of data showed no significant difference among two isolates Gnomoniopsis chinensis and the negative control (P < 0.05). However, Gnomoniopsis chinensis was still re-isolated successfully from the minor lesions caused by CFCC 52286 and CFCC 52288 and not from the negative control inoculations.
Gnomoniopsis species appear host-specific, inhabiting hosts of three families, viz. Betulaceae, Fagaceae, Rosaceae and Onagraceae (Sogonov et al. 2008;Walker et al. 2010;Visentin et al. 2012;Linaldeddu et al. 2016). Five species have been discovered from fagaceous hosts, and they are similar in conidial size (Table 2). Gnomoniopsis clavulata and G. paraclavulata were recorded on Fagus or Quercus trees (Sogonov et al. 2008). Gnomoniopsis chinensis and G. daii were discovered only from Castanea trees. It is hard to distinguish them by the currently known conidial characteristics. However, all currently known Gnomoniopsis species can be successfully distinguished by phylogenetic analysis based on ITS, tef1 and tub2. Stevanović et al. (2019) reported Gnomoniopsis idaeicola to cause blackberry canker and wilting in Serbia. With the same signs on the host bark, especially the wide, orange tendrils emerging from hosts' glaucous lenticels, Gnomoniopsis chinensis appeared to be an emerging pathogen on Castanea mollissima. Chestnut blight, caused by Cryphonectria parasitica, a notorious bark disease on chestnut trees worldwide (Rigling and Prospero 2018), can be distinguished from chestnut Gnomoniopsis canker, and the presence of mycelial fans in the cambial region. Nowadays, we have characterized the two canker pathogens on Chinese and European chestnut trees, Gnomoniopsis chinensis and G. smithogilvyi. They appear not to be very pathogenic to their native hosts, but the pathogenicity to non-native hosts is still unknown. Gnomoniopsis and Cryphonectria belong to the same fungal order Diaporthales, and are similar in some aspects. Hence, more work on these two pathogens is necessary on both Castanea mollissima and C. sativa. In addition, considering the high value of the plant genus, Castanea, and the current situation of serious commercial loss caused by various fungi, more comprehensive and detailed investigations are necessary to understand the diversity of microbes on the hosts and their functions. Table 2. Conidial size of Gnomoniopsis species from fagaceous hosts.