Characterization of Pseudofusicoccum Species from Diseased Plantation-Grown Acacia mangium, Eucalyptus spp., and Pinus massoniana in Southern China

Fungi from Pseudofusicoccum (Phyllostictaceae, Botryosphaeriales) have been reported as pathogens, endophytes, or saprophytes from various woody plants in different countries. Recently, Botryosphaeriales isolates were obtained from the dead twigs of Acacia mangium, Eucalyptus spp., Pinus massoniana, and Cunninghamia lanceolata in Guangdong, Guangxi, Hainan, and Fujian Provinces in southern China. This study aimed to understand the diversity, distribution, and virulence of these Pseudofusicoccum species on these trees. A total of 126 Pseudofusicoccum isolates were obtained, and the incidences of Pseudofusicoccum (percentage of trees that yielded Pseudofusicoccum) on A. mangium, P. massoniana, Eucalyptus spp., and C. lanceolata were 21%, 2.6%, 0.5%, and 0%, respectively. Based on the internal transcribed spacer (ITS), translation elongation factor 1-alpha (tef1), and β-tubulin (tub2) loci, 75% of the total isolates were identified as P. kimberleyense, and the remaining isolates were identified as P. violaceum. For P. kimberleyense, the majority of isolates (83%) were from A. mangium, and the rest were from P. massoniana (14%) and Eucalyptus spp. (3%). Similarly, the proportion of isolates of P. violaceum from A. mangium, P. massoniana, and Eucalyptus spp. were 84%, 13%, and 3%, respectively. Inoculation trials showed that the two species produced expected lesions on the tested seedlings of A. mangium, E. urophylla × E. grandis, and P. elliottii. This study provides fundamental information on Pseudofusicoccum associated with diseases in main plantations in southern China.


Introduction
The genus Pseudofusicoccum was proposed in 2006 based on DNA sequence data to accommodate 'Fusicoccum stromaticum' [1,2]. The status has been revised several times in recent years, and now, it is classified into Phyllostictaceae of Botryosphaeriales [3,4]. To date, nine species have been included in the genus [5]. As pathogens, endophytes, or saprophytes, species of Pseudofusicoccum have been reported from many woody plants, such as Mangifera indica, Acacia synchronica, and Eucalyptus spp., in countries including Australia, Brazil, India, South Africa, Thailand, Uruguay, and Venezuela [6]. The main diseases associated with these fungi include die-back, stem canker, and fruit rot [7][8][9].
Large plantations have been established in China, benefiting from a series of forestry programs [10]. In the subtropical and tropical areas of the country, more than 11 Mha of Cuninghamia lanceolata, 8 Mha of Pinus massoniana, and 5 Mha of Eucalyptus trees have been planted to date [11]. Acacia mangium is another popular species for plantations, but it has a relatively limited cultivation area [12].
In recent years, many diseases have been reported from these plantation trees in China, and numerous pathogens have been reported, including the fungi of Botryosphaeriaceae, Calonectria, Ceratocystis, Cryphonectriaceae, Mycosphaerellaceae, Quambalaria, and Teratosphaeriaceae, and the bacteria Ralstonia solanacearum [13][14][15][16]. Out of these, more than 20 species in Botryosphaeriales have been detected, and most of them reside in the genera Botryosphaeria,

DNA Extraction, PCR Amplification, and Sequencing
The total genomic DNA of the isolate was extracted from the mycelium of 7-day-old cultures, grown on MEA at 25 • C in the dark, using the CTAB method [19]. A total of 2 µL RNase A (10 mg/mL) was added to each DNA sample and samples were incubated at 37 • C for 1 h to remove RNA. DNA samples were checked for quality and concentration using a NanoDrop 2000 Spectrometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). For PCR amplification, the DNA samples were diluted to approximately 100 ng/µL with DNase/RNase-free ddH 2 O (Sangon Biotech Co., Ltd., Shanghai, China).
The PCR reactions were conducted in a thermocycler (BIO-RAD T100 TM , Bio-Rad Laboratories, Inc., Hercules, CA, USA). The PCR products were examined by electrophoresis in 1.5% agarose gel with 4SGelred (Sangon Biotech Co., Ltd., Shanghai, China) 1× Tris-acetate-EDTA (TAE) buffer at a constant voltage (80 V) for 40 min and visualized under UV light using a Molecular Imager Gel Doc TM XR System (Bio-Rad Laboratories, Inc., California, USA). The PCR products were sequenced in both directions by the Beijing Genomics Institution, Guangzhou, China. Sequences were inspected and manually corrected in Geneious v. 9.1.4 [23]. All of the sequences generated in this study were submitted to GenBank (http://www.ncbi.nlm.nih.gov, accessed on 22 March 2023).

Phylogenetic Analyses
Sequences of ITS, tef1, and tub2 were generated for all of the isolates obtained in this study. Based on the sequences of the three loci, the genotype of each isolate was determined, and 1-2 isolates were selected for phylogenetic analyses. Preliminary identification was conducted by sequence similarity searching using BLAST (https://blast.ncbi.nlm.nih.gov/ Blast.cgi, accessed on 8 July 2022), and the available sequences of all of the species in Pseudofusicoccum containing ex-type isolates were downloaded from NCBI for phylogenetic analyses. The sequences were aligned using the online version of MAFFT v.7 (http:// mafft.cbrc.jp/alignment/server/, accessed on 10 February 2023) [24], with the iterative refinement method (FFT-NS-i setting). The alignments were checked manually and edited in MEGA v.6.0.5 [25].
Phylogenetic analyses were conducted using maximum likelihood (ML), maximum parsimony (MP), and Bayesian inference (BI) methods for datasets of ITS, tef1, and tub2, and the combination of the three loci. ML analyses with 1000 bootstrap replicates were conducted with PhyML v.3.0 [26]. MP analyses were conducted with PAUP v.1.0b10 [27], and gaps were treated as a fifth character. BI analyses were performed with MrBayes v. 3.2.7a [28] on the CIPRES Science Gateway v. 3.3. For ML and BI analyses, the best-fit model of nucleotide substitution for each dataset was determined with jModelTest v.2.1.5 [29]. Bootstrap support values were evaluated using 1000 bootstrap replicates [30]. The phylogenetic analyses were rooted in Botryosphaeria dothidea (CBS 115476). The trees were visualized in FigTree v. 1.4.4.

Inoculation Trials
To determine the virulence of the species identified in this study, inoculation trials were conducted in a greenhouse using potted healthy seedlings of 1-year-old A. mangium, 1-year-old E. urophylla × E. grandis, and 2-year-old P. elliottii at the South China Experiment Nursery (SCEN), located in Zhanjiang, Guangdong Province, China. These seedlings were approximately 170 cm high and 2 cm in diameter at the root collar.
For each seedling, a wound (5 mm in diameter) was made on the stem (approximately 30 cm above the root collar) using a cork borer to remove the bark and expose the cambium, and the mycelial plug (5 mm diameter) from a 7-day-old culture of the selected isolate was placed into the wound with the mycelium facing the xylem. The wound with the mycelial plug was sealed with masking tape immediately to avoid contamination and desiccation. Negative control was conducted with a clean 2% MEA plug. Ten trees were inoculated for each isolate, including the negative controls. After one month, lesion lengths were measured and recorded. Re-isolations were made from the inoculated plants to fulfill Koch's postulates. One-way analysis of variance (ANOVA) was used to determine the differences in virulence among isolates utilizing SPSS v. 20 [31].

Fungal Isolation
A total of 500 samples were collected from A. mangium, 804 from Eucalyptus spp., 650 from P. massoniana, and 400 from C. lanceolata trees in southern China (Table 1). A total of 126 Pseudofusicoccum isolates identified based on ITS sequences were obtained from these trees (Tables 1 and 2). Out of these, 105 isolates (83.3%) were obtained from A. mangium, 17 isolates (13.5%) were from P. massoniana, four isolates (3.2%) were from Eucalyptus spp., and no isolates were from C. lanceolata.

Phylogenetic Analyses and Species Identification
The ITS, tef1, and tub2 loci were amplified for all 126 isolates ( Table 2). The sequence fragments were approximately 520 bp for ITS, 280 bp for tef1, and 430 bp for tub2. Sequence alignments were deposited in TreeBASE (30240). Isolates from other studies used for phylogenetic analyses were shown in Table 3. According to the phylogenetic analyses of the ITS, tef1, tub2, and the combined datasets, the isolates in this study (Group A and Group B) were most closely related to P. kimberleyense and P. violaceum ( Table 2). The sequence similarity of P. kimberleyense isolates in this study with the type of isolate (CMW 26156) were 99.42% to 99.81% for the ITS region, 98.35% to 99.01% for the tef1 gene region, and 99.08% to 100% for the tub2 gene region. The sequence similarity of P. violaceum isolates in this study with the type of isolate (CMW 22679) were 99.42% to 100% for the ITS region, 99.01% to 100% for the tef1 gene region, and 99.54% to 100% for the tub2 gene region. Although they also clustered or were closely related to P. ardesiacum and P. africanum based on the ITS dataset, they separated distinctly with the two species based on tef1, tub2, and combined datasets (Figures 1 and S1-S3). The ITS and tub2 trees showed close relationships among the isolates in this study with species of P. kimberleyense and P. violaceum, and the tef1 and combined trees provided clear results that separated isolates in Group A and Group B from the two known species (Figures 1 and S1-S3). Additionally, some isolates in this study formed an independent clade in the phylogenetic trees, but these clades had poor bootstrap values. Based on the phylogenetic analyses of the four datasets, isolates in Group A and Group B were considered the known species of P. kimberleyense and P. violaceum, respectively.

Distribution of Pseudofusicoccum
For the four plantation hosts, the incidence of Pseudofusicoccum (percentage of trees that yielded Pseudofusicoccum) was 21% on A. mangium, 2.6% on P. massoniana, 0.5% on Eucalyptus spp., and zero on C. lanceolata based on results in Table 1. Two Pseudofusicoccum

Distribution of Pseudofusicoccum
For the four plantation hosts, the incidence of Pseudofusicoccum (percentage of trees that yielded Pseudofusicoccum) was 21% on A. mangium, 2.6% on P. massoniana, 0.5% on Eucalyptus spp., and zero on C. lanceolata based on results in Table 1. Two Pseudofusicoccum species were identified from these trees, and P. kimberleyense was the dominant, comprising 75% of all of the obtained isolates, followed by P. violaceum. For isolates of P. kimberleyense, 83% were from A. mangium, 14% were from P. massoniana, and 3% were from Eucalyptus spp. For isolates of P. violaceum, 84% were from A. mangium, 13% were from P. massoniana, and 3% were from Eucalyptus spp. (Figure 2). species were identified from these trees, and P. kimberleyense was the dominant, comprising 75% of all of the obtained isolates, followed by P. violaceum. For isolates of P. kimberleyense, 83% were from A. mangium, 14% were from P. massoniana, and 3% were from Eucalyptus spp. For isolates of P. violaceum, 84% were from A. mangium, 13% were from P. massoniana, and 3% were from Eucalyptus spp. (Figure 2).

Inoculation Trials
For the two species identified, 1-3 isolates were selected for inoculations on each of the original hosts. Six isolates of the two species were used to inoculate A. mangium and E. grandis × E. urophylla, and four isolates were used to inoculate P. elliottii (Table 2). Typical lesions with a depression at the inoculation site were observed on inoculated plants, in comparison with wounds on the negative controls. Lesion and wound lengths were recorded one month after inoculation. The results showed that all of the isolates produced lesions on the tested plants, while the controls produced only small wound reactions (Figures 3 and 4). The inoculated species were re-isolated from the lesions, but never from the negative controls.

Inoculation Trials
For the two species identified, 1-3 isolates were selected for inoculations on each of the original hosts. Six isolates of the two species were used to inoculate A. mangium and E. urophylla × E. grandis, and four isolates were used to inoculate P. elliottii (Table 2). Typical lesions with a depression at the inoculation site were observed on inoculated plants, in comparison with wounds on the negative controls. Lesion and wound lengths were recorded one month after inoculation. The results showed that all of the isolates produced lesions on the tested plants, while the controls produced only small wound reactions (Figures 3 and 4). The inoculated species were re-isolated from the lesions, but never from the negative controls.
Pathogens 2023, 12, x FOR PEER REVIEW 13 of 18 species were identified from these trees, and P. kimberleyense was the dominant, comprising 75% of all of the obtained isolates, followed by P. violaceum. For isolates of P. kimberleyense, 83% were from A. mangium, 14% were from P. massoniana, and 3% were from Eucalyptus spp. For isolates of P. violaceum, 84% were from A. mangium, 13% were from P. massoniana, and 3% were from Eucalyptus spp. (Figure 2).

Inoculation Trials
For the two species identified, 1-3 isolates were selected for inoculations on each of the original hosts. Six isolates of the two species were used to inoculate A. mangium and E. grandis × E. urophylla, and four isolates were used to inoculate P. elliottii (Table 2). Typical lesions with a depression at the inoculation site were observed on inoculated plants, in comparison with wounds on the negative controls. Lesion and wound lengths were recorded one month after inoculation. The results showed that all of the isolates produced lesions on the tested plants, while the controls produced only small wound reactions (Figures 3 and 4). The inoculated species were re-isolated from the lesions, but never from the negative controls.   Overall, the lengths of lesions caused by the inoculated isolates were similar to the wounds produced by the negative controls for each of the three tree species. On A. mangium, three isolates of the two species (P. kimberleyense: CSF18503 and CSF19318, P. violaceum: CSF19320) produced lesions significantly longer than the wounds caused by the controls, while the other three isolates produced lesions not significantly different from the wounds caused by the controls (p = 0.05) ( Figure 4A). On P. elliottii, all six of the isolates produced lesions not significantly different from the wounds caused by the controls, except for isolates CSF18491 (P. kimberleyense) and CSF18430 (P. violaceum) ( Figure 4B). On E. grandis × E. urophylla, all four of the isolates produced lesions significantly longer than the wounds in the negative controls, except for isolate CSF19067 (P. kimberleyense) ( Figure  4C).

Discussion
In this study, 126 isolates of Pseudofusicoccum were obtained from the plantations of A. mangium, Eucalyptus spp., and P. massoniana from four provinces in southern China. Two species of P. kimberleyense and P. violaceum were identified based on multi-phylogenetic analyses of ITS, tef1, and tub2 loci. To our knowledge, this is the first report of Pseudofusicoccum species in China.
Genealogical concordance phylogenetic species recognition (GCPSR) provides criteria and has been applied for species delimitation for many years [39,40]. Multi-gene phylogenetic analyses without the morphological characteristics were used commonly for the identification of described species of Botryosphaeriales, including species of Pseudofusicoccum [41][42][43]. For Pseudofusicoccum species, the common loci used for phylogenetic analyses are ITS, tef1, and tub2, which can provide sufficient information to distinguish most species [2,5,32,44]. The phylogenetic analyses in this study revealed that trees based on each of the loci and a combination of the three loci were necessary for species identification, and tef1 and combined datasets were more efficient in species delimitation in this genus.
Previous studies have detected Pseudofusicoccum species in various hosts in different countries [45,46]. Out of these, P. kimberleyense was first described on Adansonia gibbosam, Acacia synchronica, Eucalyptus sp., and Ficus opposita in Australia [32,47] and also reported Overall, the lengths of lesions caused by the inoculated isolates were similar to the wounds produced by the negative controls for each of the three tree species. On A. mangium, three isolates of the two species (P. kimberleyense: CSF18503 and CSF19318, P. violaceum: CSF19320) produced lesions significantly longer than the wounds caused by the controls, while the other three isolates produced lesions not significantly different from the wounds caused by the controls (p = 0.05) ( Figure 4A). On P. elliottii, the inoculated isolates produced lesions not significantly different from the wounds caused by the controls, except for isolates CSF18491 (P. kimberleyense) and CSF18430 (P. violaceum) ( Figure 4B). On E. urophylla × E. grandis, the inoculated isolates produced lesions significantly longer than the wounds in the negative controls, except for isolate CSF19067 (P. kimberleyense) ( Figure 4C).

Discussion
In this study, 126 isolates of Pseudofusicoccum were obtained from the plantations of A. mangium, Eucalyptus spp., and P. massoniana from four provinces in southern China. Two species, P. kimberleyense and P. violaceum, were identified based on multi-phylogenetic analyses of ITS, tef1, and tub2 loci. To our knowledge, this is the first report of Pseudofusicoccum species in China.
Genealogical concordance phylogenetic species recognition (GCPSR) provides criteria and has been applied for species delimitation for many years [39,40]. Multi-gene phylogenetic analyses without the morphological characteristics were used commonly for the identification of described species of Botryosphaeriales, including species of Pseudofusicoccum [41][42][43]. For Pseudofusicoccum species, the common loci used for phylogenetic analyses are ITS, tef1, and tub2, which can provide sufficient information to distinguish most species [2,5,32,44]. The phylogenetic analyses in this study revealed that trees based on each of the loci and a combination of the three loci were necessary for species identification, and tef1 and combined datasets were more efficient in species delimitation in this genus.
Previous studies have detected Pseudofusicoccum species in various hosts in different countries [45,46]. Out of these, P. kimberleyense was first described on Adansonia gibbosam, Acacia synchronica, Eucalyptus sp., and Ficus opposita in Australia [32,47] and also reported from Carya illinoinensis in Brazil [48]. Pseudofusicoccum violaceum, first reported from Pterocarpus angolensis in South Africa [36], has been reported on Tinospora cordifolia in India [49] and Mangifera indica in Malaysia [50]. This study also showed that both were detected in A. mangium, Eucalyptus spp., and P. massoniana. A high proportion of isolates on A. mangium, compared with very rare ones on Eucalyptus spp. and P. massoniana, and no isolates on C. lanceolata in this study, revealed that species of Pseudofusicoccum associated with diseases may have a host preference in the environment.
Inoculation trials revealed that the two Pseudofusicoccum species identified in this study were virulent to the three tested hosts. This is consistent with previous studies showing that these species are also important pathogens to many hosts, including Mangifera indica [50][51][52], Syzygium malaccense [53], and Artemisia annua [9]. Although some isolates presented relatively weak virulence to hosts, such as P. adansoniae, P. ardesicum, and P. kimberleyense on baobab taproots [47], P. africanum on Mimusops caffra [33], and some P. kimberleyense and P. violaceum isolates presenting minor lesions on inoculated seedlings in this study, the co-occurrence with other botryosphaeriaceous fungi revealed that Pseudofusicoccum plays a role in disease occurrence and development [54].
The current study provides foundational data on the diversity, distribution, and virulence of Pseudofusicoccum from plantations of A. mangium, Eucalyptus spp., and P. massoniana in southern China. This study also provides evidence of the host preference of these agents. These Pseudofusicoccum species associated with stem canker and die-back indicate a new potential threat to these plantations and should not be ignored in disease management in the future.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/pathogens12040574/s1, Figure S1: Phylogenetic tree based on maximum likelihood (ML) analyses of the ITS locus for Pseudofusicoccum species. Figure S2: Phylogenetic tree based on maximum likelihood (ML) analyses the tef1 locus for Pseudofusicoccum species.

Data Availability Statement:
The DNA sequences generated in this paper were submitted to the NCBI database (https://www.ncbi.nlm.nih.gov/genbank/, accession numbers listed in the Table 2, last accessed on 22 March 2023).