﻿Identification and pathogenicity of Aurifilum species (Cryphonectriaceae, Diaporthales) on Terminalia species in Southern China

﻿Abstract The family of Cryphonectriaceae (Diaporthales) contains many important tree pathogens and the hosts are wide-ranging. Tree species of Terminalia were widely planted as ornamental trees alongside city roads and villages in southern China. Recently, stem canker and cracked bark were observed on 2–6 year old Terminalianeotaliala and T.mantaly in several nurseries in Zhanjiang City, Guangdong Province, China. Typical conidiomata of Cryphonectriaceae fungi were observed on the surface of the diseased tissue. In this study, we used DNA sequence data (ITS, BT2/BT1, TEF-1α, rpb2) and morphological characteristics to identify the strains from Terminalia trees. Our results showed that isolates obtained in this study represent two species of Aurifilum, one previously described species, A.terminali, and an unknown species, which we described as A.cerciana sp. nov. Pathogenicity tests demonstrated that both A.terminali and A.cerciana were able to infect T.neotaliala and two tested Eucalyptus clones, suggesting the potential for Aurifilum fungi to become new pathogens of Eucalyptus.


Introduction
Cryphonectriaceae is a fungal family within the order Diaporthales. This family is well-known for containing several species that are serious pathogens of trees, causing a wide range of diseases such as blight, die-back, and cankers (Gryzenhout et al. 2004(Gryzenhout et al. , 2005(Gryzenhout et al. , 2009Begoude et al. 2010;Chen et al. 2010Chen et al. , 2011Chen et al. , 2013aChen et al. , b, 2016Chen et al. , 2018Wang et al. 2018Wang et al. , 2020Roux et al. 2020). Most members of this family are easily recognizable based on the disease symptoms, as well as their distinctive yellow to orange or brown stromata and which can turn purple in 3% potassium hydroxide (KOH) and yellow in lactic acid (Gryzenhout et al. , 2009Jiang et al. 2020).
Seven of the nine families of Myrtales are commonly found in southern China, and Cryphonectriaceae has been identified as an important pathogen to Myrtales trees in previous studies (Chen et al. 2010(Chen et al. , 2011(Chen et al. , 2016Wang et al. 2018Wang et al. , 2020. Given the diverse climate and host range in southern China, there is potential for the discovery of various Cryphonectriaceae species and potential pathogens on Myrtales trees. Terminalia species are economically and ecologically important trees in southern China and are widely used for timber, medicine, and ornamental purposes (Editorial Committee of Flora of China 1988; Batawila et al. 2005;Kamtchouing et al. 2006;Angiosperm Phylogeny Group 2009). In 2019, cankers were observed on the stems of Terminalia trees during disease surveys on Myrtales trees in southern China, and fruiting structures of the fungi on the cankered stems exhibited typical Cryphonectriaceae morphological characteristics. The aims of this study were to identify the fungi isolated from these cankers based on DNA sequencing and morphological characteristics and to test their pathogenicity on Terminalia species and two widely planted E. grandis hybrid genotypes.

Disease symptoms, samples and isolations
In May 2019, disease surveys on Terminalia trees were conducted in Zhanjiang City, Guangdong Province in southern China. Sporocarps with typical characteristics of Cryphonectriaceae were observed on the surfaces of cankers on the branches, stems, and roots of Terminalia trees. In order to identify the pathogens, five experimental sites were set every 30 to 50 kilometers. Diseased bark pieces, branches, twigs, and roots bearing fruiting structures were collected and transported to the laboratory. The fruiting structures were incised using a sterile scalpel blade under a stereoscopic microscope. The spore masses were then transferred to 2% (v/v) malt extract agar (MEA) and incubated at room temperature for three to five days until colonies developed. The pure cultures were obtained by transferring single hyphal tips from the colonies to 2% MEA plates and incubated at room temperature for 7-10 days. The pure cultures are stored in the culture collection (CSF) at the Research Institute of Fast-Growing Trees (RIFT) (previous institution: China Eucalypt Research Centre, CERC), Chinese Academy of Forestry (CAF) in Zhanjiang, Guangdong Province, China.

DNA extraction, polymerase chain reaction (PCR) amplification and sequencing
Representative isolates were selected for DNA sequence analyses, and actively growing mycelium on MEA cultures grown for one week at room temperature was scraped using a sterilized scalpel and transferred into 2.0 mL Eppendorf tubes. Total genomic DNA was extracted using the cetyltrimethylammonium bromide (CTAB) method described by Van Burik et al. (1998). The extracted DNA was dissolved in 30 μL TE buffer, and the concentration was measured using a Nano-Drop 2000 spectrometer (Thermo Fisher Scientific, Waltham, Massachusetts).
All amplified products were sequenced in both directions using the same primers that were used for the PCR amplification. Sequence reactions were performed by the Beijing Genomics Institute of Guangzhou, China. The nucleotide sequences were edited using Geneious 7.1.8 software. The sequences obtained in this study were submitted to GenBank (http://www.ncbi.nlm.nih.gov).

Phylogenetic analysis
The preliminary identities of the isolates sequenced in this study were obtained by conducting a standard nucleotide BLAST search using the ITS, BT2, and BT1 sequences. The BLAST results showed that the isolates collected in this study were mainly grouped in the genus Aurifilum. Phylogenetic analyses for strains identification in the current study were conducted for both genetic and species identification.
To determine the placement of Aurifilum species, two represent strains in this study were first determined by conducting phylogenetic analyses within Cryphonectriaceae species (Table 1) on combined datasets for the ITS and BT2/BT1 regions. Then, the strains in the Aurifilum genus were further analyzed and identified using separate and combined datasets for the ITS, BT2/BT1, TEF-1α, and rpb2 regions. Sequences of the Aurifilum isolates collected in this study and those from NCBI were aligned using MAFFT 7 (http://mafft.cbrc.jp/alignment/server) with the interactive refinement method (FFT-NS-i) setting (Katoh and Standley 2013). Then they were manually edited in MEGA X.
The taxonomic positions of two methods were used for phylogenetic analyses. Maximum parsimony (MP) analyses were performed using PAUP v. 4.0 b10 (Swofford 2003) and maximum likelihood (ML) analyses were conducted with PhyML v. 3.0 (Guindon and Gascuel 2003).
For MP analyses, gaps were treated as a fifth character, and characters were unordered and of equal weight with 1,000 random addition replicates. A partition homogeneity test (PHT) using PAUP v. 4.0 b10 (Swofford 2003) was conducted to determine whether data for the four genes could be combined. The most parsimonious trees were obtained using the heuristic search option with stepwise addition, tree bisection, and reconstruction branch swapping. MAXTREES was set to 5,000 and zero-length branches collapsed. A bootstrap analysis (50% majority rule, 1,000 replicates) was carried out to determine statistical support for internal nodes in trees. Tree length (TL), consistency index (CI), retention index (RI) and homoplasy index (HI) were used to assess phylogenetic trees (Hillis and Huelsenbeck 1992).    For ML analyses, the best nucleotide substitution model for each dataset was established using jModeltest v. 2.1.5 (Posada 2008). In PhyML, the maximum number of retained trees was set to 1,000 and nodal support was determined by non-parametric bootstrapping with 1,000 replicates. For both MP and ML analyses, the phylogenetic trees were viewed using MEGA v. 6.0.

Morphology
The representative isolates identified as the new species by DNA sequence analysis were grown on 2% water ager (WA), to which sterilized freshly cut branch sections (0.5-1 cm diam. 4-5 cm length) of Eucalyptus urophylla × E. grandis (CEPT53) branch sections were added. These fungi with branch sections on 2% WA were incubated at room temperature for 6-8 wks until fruiting structures emerged. Representative cultures are maintained in the China General Microbiological Culture Collection Centre (CGMCC), Beijing, China. Isolates linked to the type specimens connected to representative isolates were deposited in the mycological fungarium of the Institute of Microbiology, Chinese Academy of Sciences (HMAS), Beijing, China, and the Collection of Central South Forestry Fungi of China (CSFF), Guangdong Province, China.
The structures that emerged on the surface of the Eucalyptus branches were mounted in one drop of 85% lactic acid on glass slides under a dissecting microscope and then embedded in Leica Bio-systems Tissue Freezing Medium (Leica Biosystems nussloch GmbH, Nussloch, Germany) and sectioned (6 μm thick) using a Microtome Cryostat Microm HM550 (Microm International GmbH, Thermo Fisher Scientific, Walldorf, Germany) at -20 °C. Conidiophores, conidiogenous cells, and conidia were measured after crushing the sporocarps on microscope slides in sterilized water. For the holotype specimens, 50 measurements were performed for each morphological feature, and 30 measurements per character were made for the remaining specimens.
Isolates identified as new species were selected for studying culture characteristics. After the isolates were grown for 7 days on 2% MEA, a 5 mm plug was  Rayner (1970).

Pathogenicity tests
In this study, inoculations were conducted on two different Eucalyptus hybrid genotypes (CEPT46 and CEPT53) and T. neotaliala to understand the pathogenicity on Eucalyptus plantations and to fulfill Koch's postulates. The selected isolates were grown on 2% MEA at 25 °C for 10 days before inoculation. Each selected isolate was inoculated on 10 seedlings or branches of each inoculated tree, and 10 additional seedlings or branches were inoculated with sterile MEA plugs to serve as negative controls. The inoculations were conducted in August 2019, and the results were evaluated after 7 weeks by measuring the lengths of the lesions on the cambium. Inoculations were conducted on T. mantaly and two widely planted E. grandis hybrid genotype (CEPT46, CEPT53) to fulfill Koch's postulates and understand the pathogenicity on Eucalyptus plantations. The selected isolates were grown on 2% MEA at 25 °C for 10 d before inoculation. Each of the selected isolates was inoculated on 10 seedlings or branches of each selected tree variety, and 10 additional seedlings or branches were inoculated with sterile MEA plugs to serve as negative controls. The inoculations on seedlings of two 1-year-old Eucalyptus hybrid genotypes were conducted in the glasshouse, and the inoculations on branches of 10-year-old T. mantaly were conducted in the field. The inoculations method followed Chen et al. (2010Chen et al. ( , 2013b. Inoculations were conducted in August 2019 and the results were evaluated after 7 weeks by measuring the lengths (mm) of the lesions on the cambium. For re-isolations, small pieces of discolored xylem from the edges of the resultant lesions were cut and placed on 2% MEA at room temperature. Re-isolations of all seedlings/branches inoculated as negative controls and from four randomly selected trees per isolate were conducted. The identities of the re-isolated fungi were confirmed by morphological comparisons. The inoculation results were analyzed using SPSS Statistics 26 software (BM Corp., Armonk, NY, USA) by one-way analysis of variance (ANOVA).

Isolation
Diseased samples from 14 trees were collected from three sites (20190523-1, 20190525-2, 20190525-3) of T. neotaliala (Fig. 1A) nurseries, and two sites (20190525-1, 20190525-4) of T. mantaly (Fig. 1B) nurseries (Table 2). In the surveyed sites, 10%-25% of Terminalia trees were infected. Cankers with stro-mata on the main stem bark surface, which often resulted in tree death, were observed on two to five-year-old T. neotaliala trees (Fig. 1C). Obvious orange conidiomata were observed on the branches and twigs of three-year-old T. mantaly trees (Fig. 1D, E). Developing lesions were observed on the main stem of T. neotaliala and resulting in bark depression (Fig. 1F) and xylem necrosis (Fig. 1G). Orange fruiting structures even presented on the barks of the main stem base (Fig. 1H) and roots (Fig. 1I). The fruiting structures on T. neotaliala and T. mantaly displayed the typical morphological characteristics of Cryphonectriaceae (Gryzenhout et al. 2009;Wang et al. 2020). Isolates obtained from the asexual fruiting structures on MEA were white when young and turned yellow with age, and the isolates on MEA exhibited typical morphological characteristics of Cryphonectriaceae. Twenty isolates from both T. neotaliala and T. mantaly in the five sampled nurseries were isolated and sequenced for further studies (Table 2).

Phylogenetic analysis
Phylogenetic analyses indicated that all of the Cryphonectriaceae genera formed independent phylogenetic clades with high bootstrap values (ML > 77%, MP > 100%) both in the ML and MP analyses, with the exception of Aurifilum, and strains sequenced in this study formed sub-clades (Fig. 2). The partition homogeneity test (PHT), comparing the combined ITS and BT2/BT1 loci dataset generated a value of P was 0.68, indicating some incongruence in the dataset of the four loci, and the accuracy of the combined data suffered relative to the individual partitions (Huelsenbeck et al. 1996;Cunningham 1997).
Further species analyses selected twenty-four Aurifilum isolates (Table 2). Based on the sequences of ITS, BT2/BT1, TEF-1α, rpb2 sequences, four genotypes were generated for the 20 isolates sequenced in this study (Table 2). Sequences for two ex-type specimen strains and other of two Aurifilum species related to isolates obtained in this study were downloaded from GenBank (Table 1). Celoporthe cerciana (CERC9128) was used as an outgroup taxon. The partition homogeneity test (PHT), comparing the combined ITS, BT2/BT1, TEF-1α and rpb2 loci dataset generated a value of P was 1, indicating some incongruence in the dataset of the four loci, and the accuracy of the combined data suffered relative to the individual partitions (Huelsenbeck et al. 1996;Cunningham 1997). Although the P value was high, the sequence of four loci was combined and subjected to phylogenetic analyses. All sequences obtained for the isolates of Aurifilum in this study were deposited in GeneBank ( Table 2). The number of taxa and characters in each of the datasets, and the summary of the most important parameters applied in the MP and ML analyses, are presented in Table 3. The six datasets were deposited in TreeBASE (http://purl.org/phylo/treebase/phylows/study/TB2:S30284?x-access-code=cf2a0ef843604b8fa4301eced72cec7f&format=html,30284).
For each of the six datasets, the MP and ML analyses generated trees with generally consistent topologies and phylogenetic relationships among taxa. Among the trees generated by the Aurifilum spp. single loci dataset, the BT2/ BT1, rpb2 show that 20 isolates obtained in this study mainly grouped into two clades, one clade contained nine isolates cluster into a lineage with A. terminali, the other 11 isolates clade formed a novel monophyletic lineage that was distinct from any known Aurifilum sp., and was supported by high bootstrap values in these gene trees (Fig. 3B-D).    Among the BT2/BT1 trees, isolate CSF16343, CSF16387, CSF16388 grouped into the lineage with A. terminali, and among the rpb2 tree, isolates CSF16351, CSF16352 grouped into the novel lineage, formed a single independent branch but the bootstraps value within the clades were not significant (Fig. 3B, D), which suggests that these differences reflect intraspecific rather than interspecific variation. The combined ITS, BT2/BT1, TEF-1α and rpb2 tree (Fig. 3E) indicated that the isolates grouped into novel lineage are putative undescribed species of Aurifilum (bootstrap values of the combined dataset, ML and MP: 96 and 100%).

Morphology and taxonomy
Based on phylogenetic analyses and morphology characteristics, the isolates from Terminalia trees in southern China represent two distinct species in Aurifilum. Isolates CSF16295, CSF16309, CSF16310, CSF16343, CSF16356, CSF16377, CSF16380, CSF16387, CSF16388 in phylogenetic cluster with A. terminali (Fig. 3B-E), and isolates CSF16343, CSF16387, CSF16388 appear a branch in BT2/BT1, rpb2, and combine trees (Fig. 3B, D, E) in this cluster, was finally identified as A. terminali. The isolates in the other cluster present a novel species in Aurifilum, here named as Aurifilum cerciana sp. nov. (Fig. 3); this unknown species was described as follows:  Bootstrap value lower than 70% are marked with *, and absent analysis value are marked with -. Isolates representing A. cerciana are in shade, and isolates obtained in this study are numbered followed CSF. Celoporthe circiana (CERC9128) was used as outgroup taxon.  Fig. 4 Etymology. the name refers to China Eucalypt Research Centre (CERC), the former institution of the Research Institute of Fast-Growing Trees (RIFT), which served as the identification site for this study on Terminalia trees disease caused by Aurifilum spp.
A. cerciana could also be distinguished from A. terminali and A. marmelostoma by growth characteristics in culture. The optimal growth temperature of A. cerciana is 35 °C, whereas A. terminali grows relatively slowly at this temperature and no growth is observed for A. marmelostoma (Begoude et al. 2010;Wang et al. 2020).

Pathogenicity tests.
Eight isolates representing the two species of Aurifilum identified in this study were used to inoculate seedlings of two Eucalyptus hybrid genotypes, and branches of T. neotaliala. These include four isolates in A. terminali and A. cerciana, respectively (Table 2). Seedling stems or tree branches inoculated with Aurifilum isolates exhibited lesions, whereas the control group only showed wounds without any lesions. (Fig. 5). The lesions produced by Aurifilum species on T. neotaliala and Eucalyptus clones CEPT53 were significantly longer than the wounds on the controls (P < 0.05), whereas for the Eucalyptus clones CEPT46, the lesions produced by Aurifilum species were not significantly different (Fig. 5). The overall data revealed that A. cerciana and A. terminali have similar pathogenicity (Fig. 5). The overall data further showed that CEPT53 is more susceptible than CEPT46 to Aurifilum spp. (Fig. 5B). Yellow or orange fruiting structures and cankers were produced on the bark of inoculated trees within 7 weeks; these structures displayed similar characteristics of conidiomata on the Terminalia trees in the field and the re-isolated fungi from lesions share the same culture morphology with the Aurifilum fungi originally from the Terminalia trees in the nursery. The inoculated Aurifilum fungi were successfully re-isolated from the lesions but not from the control, indicating that the Koch's postulates had been fulfilled.

Discussion
In this study, many Aurifilum isolates were obtained from diseased Terminalia trees in Southern China, and two species of four genotypes belonging to Aurifilum were identified from two species of Terminalia. Including the new taxon, A. cerciana sp. nov., there are fifty-seven taxa in the Cryphonectriaceae.
In the genus Aurifilum, A. marmelostoma was the first described species, which was isolated from the bark of native T. ivorensis and the dead branches of non-native T. mantaly in Cameroon (Begoude et al. 2010), and the A. terminali, the second identified species, was isolated from non-native T. neotaliala in southern China (Wang et al. 2020). In the present study, a new species, A. cerciana was isolated from non-native T. neotaliala and T. mantaly, Figure 5. Column chart showing average lesion lengths (mm) produced by each isolate of Aurifilum on the branches of T. neotaliala (left) and two Eucalyptus hybrid genotypes (right). Eight isolates of Aurifilum were used. Vertical bars represent the standard error of the means. Different letters above the bars indicate treatments that were statistically significantly different (P = 0.05). and a previously known species, A. terminali was isolated from T. mantaly too. The species T. mantaly was a newly reported host for A. terminali. Our results indicated that the Aurifilum species are widely distributed on non-native Terminalia trees in southern China, which is consistent with the previous hypothesis of Wang et al. (2020).
Members of the Cryphonectriaceae are well known to occur on Myrtales in Southern China. Prior to this study, six genera, including Aurifilum, Celoporthe, Chrysoporthe, Chrysomorbus, Corticimorbus, Parvosmorbus were reported infecting trees in Combretaceae, Lythraceae, Melastomataceae and Myrtaceae (All Myrtales) in southern China (Chen et al. 2010(Chen et al. , 2011(Chen et al. , 2016Wang et al. 2020). Although the diversity of Cryphonectriaceae in Myrtales has been extensively studied in recent years (Chen et al. 2010(Chen et al. , 2011(Chen et al. , 2013a(Chen et al. , b, 2016a(Chen et al. , b, 2018Wang et al. 2018Wang et al. , 2020Huang et al. 2022), there is still a need for further investigation into its diversity, geographical distribution, and host range in China (Wingfield et al. 2015).
Pathogenicity test showed that all tested Aurifilum isolates were pathogenic to mature T. neotaliala and E. grandis hybrid genotypes of CEPT53 and CEPT46 seedlings. To clarify the threat of these pathogens to Eucalyptus plantations, further inoculations on mature Eucalyptus in the field should be conducted. Variations in pathogenicity among different individuals of the same species have been observed, with some strains showing stronger pathogenicity in different hosts. This phenomenon has also been observed in previous studies (Chen et al. 2010(Chen et al. , 2011(Chen et al. , 2013a2016a, b, 2018Wang et al. 2018Wang et al. , 2020, and further comparison of the genetic features of these individual exhibiting differences in pathogenicity may help reveal the pathogenic mechanisms of the pathogen.