The branches of 3 S. apetala trees with canker lesions with conidiomata were collected and isolations were conducted in the laboratory for 2 types of infections. For cankered lesions on the bark, the branches were cleaned with tap water and small pieces of bark (sized approximately 2 mm × 2 mm) were cut from the junction of the diseased and healthy portions. These small pieces were disinfected in 75% ethanol for 5 s and transferred to a 3% sodium hypochlorite (NaClO) solution for 2 min. Then, the samples were washed thrice with sterile water and inoculated on the surface of potato dextrose agar (PDA) plates. For branches with conidiomata, single conidial isolates were obtained by removing the spore masses into axenic water in order to obtain the suspension and spread the suspension on to the surface of PDA plates for isolation (Jiang et al. 2018b).

The morphological features of the pathogenic fungi were observed on diseased plant tissues following Fan et al. (2018). Species identification was performed based on the morphological characters of the sporocarp produced on the diseased spots. First, cross-sections were made using a double-edge blade. Then, the morphological characters of sporocarp were recorded. For example, the size of conidiomata and locules were measured by using a dissecting stereomicroscope, while the shape and size of conidiophores and conidia were determined using a Leica compound microscope (LM, DM 2500). Finally, sporocarps were sectioned using a hand-operated blade and more than 50 spores were selected randomly and measured under the Leica compound microscope.

Aerial mycelium of fungi grown on PDA (for 7 days at 25ºC) was used to extract the genomic DNA. The DNA extraction was performed by the modified CTAB method (Doyle and Doyle 1990). The internal transcribed spacer of rDNA (ITS) was amplified with the primers ITS1 and ITS4 (White et al. 1990). The translation elongation factor-1 alpha (TEF) was amplified with the primers EF1-688F and EF1-1251R (Alves et al. 2008). Two regions within the β-tubulin (BT1/BT2) gene were amplified with the primers Bt1a/Bt1b and Bt2a/Bt2b (Glass and Donaldson 1995). PCR amplification and sequencing were performed following the protocol of Wang et al. (2018). The PCR amplification products were estimated visually by electrophoresis in 2% agarose gel at 60 V for 90 min. DNA sequencing was performed using an ABI PRISM® 3730XL DNA Analyzer with the BigDye® Terminater Kit v.3.1 (Invitrogen) at the Shanghai Invitrogen Biological Technology Company Limited (Beijing, China). Two isolates were detected for each species.

The 10 new sequences generated in this study and the reference sequences of Celoporthe and Cytospora isolates selected from recent studies, were included in the phylogenetic analyses (TW). These sequences were aligned with MAFFT v.7 (Katoh and Toh 2010) and manually adjusted. For Celoporthe, phylogenetic analyses were performed, based on the combined ITS-BT1-BT2-TEF sequences by PAUP v. 4.0b10 (Swofford and Sullivan 2003) for Maximum Parsimony (MP), PhyML v.3.0 (Guindon et al. 2010) for Maximum Likelihood (ML) and MrBayes v.3.1.2 (Ronquist and Huelsenbeck 2003) for Bayesian Inference (BI), respectively. For Cytospora, ITS sequences were used to conduct phylogenetic analyses using the same software. Information on the isolates, the genes sequenced and GenBank accessions used in this study are all included in Suppl. material 1.

For pathogenicity trials, the 2 isolates TLY1-15 (Celoporthe guangdongensis) and TLY2-42 (Cytospora rhizophorae) were randomly selected for the inoculation studies. The inoculations were performed on the branches of healthy S. apetala trees as per the the methods described by Chen et al. (2011). Briefly, the detached branches (aged: 1–2-years-old, approximately 1.0-cm diameter) from healthy S. apetala trees were selected and cut into 20-cm-long pieces. A total of 90 fresh branches were used for the pathogenicity tests and 45 branches were cut and inoculated with each of the two isolates or sterile PDA, respectively. The other branches were scalded and inoculated with the same 2 isolates or sterile PDA. The inoculated branches were sealed in Petri dishes with a gauze immersed in sterile water and maintained in a greenhouse at 25°C.

After 2 weeks, some symptoms were detected on the surface of the inoculated branches. The lesion sizes in the cambium were measured from all experimental and control groups. Re-isolations were performed on PDA and the re-isolation cultures were identified by DNA testing.

Differences in the lesion sizes between the isolates and negative controls were analyzed by one-way analysis of variance (ANOVA), followed by least significant difference (LSD) tests. Statistical analysis was performed by using the R v.3.4.3 software and considered to be significant at P < 0.05.

Three isolations (iTLY1-18 inclusive) obtained from the branches of S. apetala with canker lesions and 4 isolations (TLY1-15 inclusive) from branches with conidiomata had the same cultural phenotypes on PDA. Another 4 isolations (TLY1-13 and TLY2-42 inclusive) collected from the branches with similar conidiomata showed the same cultural phenotypes on PDA. Finally, we selected the TLY1-18 and TLY1-15, TLY1-13 and TLY2-42 for molecular phylogeny.

In the genus Celoporthe, the combined ITS, BT1, BT2 and TEF alignment contained 23 sequences (including 2 outgroups) and 1684 characters including alignment gaps; of which 1387 were parsimony informative, 159 were variable and parsimony uninformative, and 138 were constant. The MP analysis revealed 2 equally most-parsimonious trees; the first tree (TL = 367, CI = 0.907, RI = 0.934, RC = 0.848) is shown in Fig. 3. The topology of the phylogenetic trees obtained from ML and Bayes were similar to that of the MP tree. The Celoporthe isolates from the present study cluster in a clade were found to be closely related to the ex-type of C. guangdongensis CMW 12750 (Fig. 3).

In the genus Cytospora, the ITS alignment contained 24 sequences (including one outgroup) and 620 characters including alignment gaps; of which 442 were parsimony informative, 71 were variable and parsimony uninformative and 107 were constant. The MP analysis resulted in 21 equally most-parsimonious trees; the first tree (TL = 251, CI = 0.853, RI = 0.908, RC = 0.774) is shown in Fig. 5.The topology of phylogenetic trees obtained from ML and Bayes were similar to that of the MP tree. The Cytospora isolates from the present study and 2 C. rhizophorae strains were clustered in a supported clade (Fig. 5).

The isolates of Celoporthe guangdongensis, TLY1-15 on S. apetala in the greenhouse showed pathogenicity, but no pathogenicity was detected in any of the inoculations with the blank control or Cytospora rhizophorae within 6 weeks. Two weeks after inoculation, bark lesion was shown on the scalded branches inoculated with C. guangdongensis. Subsequently, the lesion was also exhibited on the wound of S. apetala trees branches treated with C. guangdongensis (Fig. 6). The two treatments inoculated with C. guangdongensis on the branches of S. apetala produced significantly longer lesions as compared with that in the control after 4 weeks (P = 0.05) (Fig. 7). Moreover, yellow or orange fruiting structures and cankers were produced on the barks inoculated with C. guangdongensis after 4 weeks (Suppl. material 2). However, all treatments inoculated with C. rhizophorae and the blank control produced no bark lesions. The cultural phenotypes and ITS sequences of re-isolations were the same as the tested strains.

Figure 6.  

Lesions resulting from inoculation of Celoporthe guangdongensis and Cytospora rhizophorae on to Sonneratia apetala branches and wound response on the negative controls; negative control (a), Celoporthe guangdongensis (b), Cytospora rhizophorae (c). Line 1, inoculated on to incised wound; line 2, inoculated on to scald wounds.

Figure 7.  

Histogram showing the average lesion area (mm²) resulting from inoculations of Sonneratia apetala with Celoporthe guangdongensis (TLY1-15) and Cytospora rhizophorae (TLY2-42). Treatment 1 inoculated on to incised wound; Treatment 2 inoculated on to scald wounds. Bars represent 95% confidence limits for each treatment. Different letters above the bars indicate treatments that statistically were significantly different (P = 0.05).