Wide Distribution and Intraspecies Diversity in the Pathogenicity of Calonectria in Soil from Eucalyptus Plantations in Southern Guangxi of China

Eucalyptus spp. are extensively cultivated in southern China because of their adaptability and versatile timber production. Calonectria leaf blight caused by Calonectria species is considered a major threat to Eucalyptus trees planted in China. The GuangXi Zhuang Autonomous Region is the provincial region with the largest distribution of Eucalyptus plantations in China. The present study aimed to expound the species diversity and pathogenicity of Calonectria isolates obtained from the soil of Eucalyptus plantations in GuangXi. A total of 188 Calonectria isolates were recovered from the soil located close to Eucalyptus trees, and the isolates were identified based on the DNA sequence comparisons of the four partial regions of the translation elongation factor 1-alpha (tef1), β-tubulin (tub2), calmodulin (cmdA), and histone H3 (his3) genes. The isolates were identified as Calonectria aconidialis (74.5%), C. hongkongensis (21.3%), C. pseudoreteaudii (2.1%), C. kyotensis (1.6%), and C. chinensis (0.5%). The inoculation results indicated that 40 isolates representing five Calonectria species were pathogenic to the three Eucalyptus genotypes. Two inoculated experiments consistently showed that the longest lesions were produced by the isolates of C. aconidialis. Some isolates of C. aconidialis, C. hongkongensis, and C. kyotensis produced significantly longer lesions than the positive controls, but not the isolates of C. pseudoreteaudii or C. chinensis. These results indicated that Calonectria isolated from the soil may pose a threat to Eucalyptus plantations. Some Calonectria isolates of the same species differed significantly in their virulence in the tested Eucalyptus genotypes. The resistance of different Eucalyptus genotypes to Calonectria isolates within the same species was inconsistent. The inoculation results in this study suggested that many Calonectria isolates in each species had different levels of pathogenicity, and many Eucalyptus genotypes need to be tested to select disease-resistant Eucalyptus genetic materials in the future. The results of the present study enhance our knowledge of species diversity and the potential damage caused by Calonectria in the soil of Eucalyptus plantations. Our results also provide new insights into the breeding of disease-resistant Eucalyptus genotypes for controlling Calonectria leaf blight in China in the future.


Introduction
Eucalyptus L'Hér. (Myrtaceae Juss., Myrtales Juss. ex Bercht. and J.Presl), due to its rapid growth, robust adaptability, and broad applications, is extensively planted in tropical and subtropical regions in China [1]. Eucalyptus was originally introduced to China in 1890 as an ornamental plant [1]. The area covered by Eucalyptus plantations has increased exponentially, from 0.46 million hm 2 in 1986 to 5.46 million hm 2 in 2018 [2]. In China, Eucalyptus plantations are distributed mainly in GuangXi, GuangDong, YunNan, FuJian, SiChuan, and HaiNan Provinces (or Autonomous Regions). The GuangXi Zhuang Autonomous Region is the provincial region with the largest distribution of Eucalyptus plantations in this country [1]. The area of Eucalyptus plantations in GuangXi is 2.56 million hm 2 , which is 46.83% of the total area of Eucalyptus plantations in the country [3].
Several Calonectria species isolated from blighted Eucalyptus leaves and soil in Eucalyptus plantations in China were pathogenic to the tested Eucalyptus genotypes [7][8][9]28,29]. Some of these species were acquired from diseased Eucalyptus tissues (leaves and branches) and soil close to these trees. In the present study, soil samples were obtained from Eucalyptus plantations in GuangXi. The purposes of this study were to (i) expound the species diversity of Calonectria isolated from these soil samples, and (ii) clarify the pathogenicity of Calonectria species on different Eucalyptus genotypes.

Sample Site, Collection, and Fungal Isolation
Soil samples were collected from Eucalyptus plantations between July and August 2019 in GuangXi, southern China. These plantations were located at seven sampling sites across four regions, BeiHai, QinZhou, FangchengGang, and ChongZuo Region ( Figure 1, Table 1). The soil in the 3-5-year-old Eucalyptus plantations was relatively moist with thick layers of leaf litter. The upper 0-20 cm of the soil was extracted by removing the thick layers of leaf litter. Fifty-three to sixty-nine soil samples were randomly collected from each sample site (Table 1). The soil samples were first placed in plastic bags to maintain humidity and temperature and then transferred to a laboratory for fungal isolation and further molecular studies.
To induce Calonectria isolates, distilled water was utilized to moisten the soil samples in plastic cups. Medicago sativa L. (alfalfa) seeds were surface disinfested in 75% ethanol for 30 s and washed with distilled water. They were then placed on the surface of the moistened soil in plastic cups, as described by Crous [11]. The sampling cups with soil and alfalfa seeds were incubated at 25 • C under 12 h of daylight and 12 h of darkness. After 7 d, the sampling cups with soil and germinating alfalfa seedlings were observed under a dissection microscope. Calonectria isolates were distinguished from other fungi based on the typical morphological characteristics of conidiophores, macroconidia, and vesicles [11,18,30]. A single conidium was transferred from the conidiophores of Calonectria to a 2% (v/v) malt extract agar (MEA) (20 g of malt extract powder and 20 g of agar powder per liter of water) using sterile needles under a stereoscopic microscope. For each soil sample, a culture of one morphologically similar Calonectria isolate was retained for further studies. The obtained cultures were deposited in the culture collection (CSF) located at the Research Institute of Fast-growing Trees (RIFT) of the Chinese Academy of Forestry (CAF) in ZhanJiang, GuangDong Province, China.  To induce Calonectria isolates, distilled water was utilized to moisten the soil samples in plastic cups. Medicago sativa L. (alfalfa) seeds were surface disinfested in 75% ethanol for 30 s and washed with distilled water. They were then placed on the surface of the moistened soil in plastic cups, as described by Crous [11]. The sampling cups with soil and alfalfa seeds were incubated at 25 °C under 12 h of daylight and 12 h of darkness. After 7 d, the sampling cups with soil and germinating alfalfa seedlings were observed under a dissection microscope. Calonectria isolates were distinguished from other fungi based on the typical morphological characteristics of conidiophores, macroconidia, and vesicles [11,18,30]. A single conidium was transferred from the conidiophores of Calonectria to a 2% (v/v) malt extract agar (MEA) (20 g of malt extract powder and 20 g of agar powder per liter of water) using sterile needles under a stereoscopic microscope. For each soil sample, a culture of one morphologically similar Calonectria isolate was retained for further studies. The obtained cultures were deposited in the culture collection (CSF) located at the Research Institute of Fast-growing Trees (RIFT) of the Chinese Academy of Forestry (CAF) in ZhanJiang, GuangDong Province, China.

DNA Extraction, PCR Amplification, and Sequencing
The DNA was extracted after the isolates were grown on MEA for 7-10 days. Mycelia were carefully scraped from the surface of the MEA culture medium using a sterilized scalpel and transferred to a 2 mL Eppendorf tube. Total genomic DNA was extracted according to "Extraction method 5: grinding and CTAB" protocols described by van Burik et al. [31]. The extracted DNA was dissolved in 30 µL of TE buffer (1 M Tris-HCl and 0.5 M EDTA, pH 8.0), and then 3 µL of RNase (10 mg/mL) was added at 37 °C for 1 h to degrade the RNA. In the final step, a Nano-Drop 2000 spectrometer (Thermo Fisher Sci-

DNA Extraction, PCR Amplification, and Sequencing
The DNA was extracted after the isolates were grown on MEA for 7-10 days. Mycelia were carefully scraped from the surface of the MEA culture medium using a sterilized scalpel and transferred to a 2 mL Eppendorf tube. Total genomic DNA was extracted according to "Extraction method 5: grinding and CTAB" protocols described by van Burik et al. [31]. The extracted DNA was dissolved in 30 µL of TE buffer (1 M Tris-HCl and 0.5 M EDTA, pH 8.0), and then 3 µL of RNase (10 mg/mL) was added at 37 • C for 1 h to degrade the RNA. In the final step, a Nano-Drop 2000 spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) was used to measure the DNA concentration.
All the PCR products were sequenced in both the forward and reverse directions of each primer pair at the Beijing Genomics Institute, GuangZhou, China. The sequences were manually edited using MEGA v. 6.0 software [36] and then submitted to GenBank (https://www.ncbi.nlm.nih.gov, accessed on 8 March 2023).

Phylogenetic Analyses
To preliminarily identify the isolates, a standard nucleotide BLAST search was performed using the tef1, tub2, cmdA, and his3 sequences. The sequences of the available species in the relevant species complexes were downloaded from NCBI for sequence comparisons and phylogenetic analyses. The alignment of sequences for each of the tef1, tub2, cmdA, and his3 gene regions, as well as the combination of these four gene regions, was performed online using MAFFT v. 7 (http://mafft.cbrc.jp/alignment/server/, accessed on 8 March 2023) with alignment strategy FFT-NS-i (slow; interactive refinement method) [37]. The manual sequence adjustment was performed using MEGA v. 7 software [38].
The maximum likelihood (ML) and Bayesian inference (BI) methods were used for the phylogenetic analysis of the sequence datasets of each of the four gene regions and the combination of these regions. The optimal models of the five sequence datasets for BI analyses were determined using the jModelTest v. 2.1.5 [39]. ML analyses were performed using RaxML v. 8.2.12 [40] on the CIPRES Science Gateway v. 3.3, with the default GTR substitution matrix and 1000 bootstrap runs. The software MrBayes v. 3.2.7 [41] was used for BI analyses with CIPRES Science Gateway v. 3.3. Four Markov chain Monte Carlo (MCMC) chains were executed from a random starting tree for five million generations, and the trees were sampled every 100th generation. The first 25% of the trees were discarded as burn-in, and the rest of the trees were used to confirm the posterior probabilities. Phylogenetic trees were viewed using MEGA v. 7 [38] and FigTree v 1.4.2 for ML and BI trees, respectively. The sequence data for CBS 109167 and CBS 109168 (Curvicladiella cignea Decock and Crous) were treated as outgroups [22].

Pathogenicity Tests
Representative isolates of each Calonectria species identified in this study were selected for inoculation trials. Three Eucalyptus genotypes were selected for inoculation, E. urophylla S. T. Blake × E. tereticornis Sm. hybrid genotype CEPT1900 and E. urophylla × E. grandis W. Hill hybrid genotypes CEPT1901 and CEPT1902. All inoculated seedlings were similar in size, 3 months old, and approximately 40 cm in height.
Inoculation with mycelial plugs was performed as described by Wu and Chen [9]. For each Eucalyptus genotype, 10 mycelial plugs (5 mm diameter) from 7-day-old MEA cultures of each isolate were inoculated on the abaxial surface of the unwounded leaves of three Eucalyptus seedlings. Ten leaves from three different Eucalyptus seedlings treated with sterile MEA plugs were used as negative controls. The highly pathogenic Calonectria pseudoreteaudii L. Lombard, M.J. Wingf. and Crous, isolate CSF13317 of two Eucalyptus hybrid genotypes, E. urophylla × E. grandis genotype CEPT1878 and E. urophylla × E. tereticornis genotype CEPT1879, as confirmed in a previous study, was used as a positive control [29]. To ensure sufficient humidity for infection development, all Eucalyptus seedlings were placed in moist plastic chambers and maintained under stable climatic conditions (temperature 25-26 • C; humidity 60-70%) for three days. The plastic chambers were removed after three days. To measure the lesion length of each leaf, two diameter measurements of each lesion perpendicular to each other were conducted for each leaf, and the average lesion diameter was computed. The entire experiment was repeated using an identical methodology. The inoculations were conducted in July 2022 at the South China Experimental Nursery (SCEN), located in ZhanJiang, GuangDong Province, China.
To verify Koch's postulates, re-isolations were conducted. Small pieces of discolored leaf tissue (approximately 0.04 cm 2 ) from the periphery of the generated lesions were cut and placed on a 2% MEA at room temperature. For each inoculated isolate, four leaves of each Eucalyptus genotype were randomly selected, and all the leaves inoculated as positive and negative controls were re-isolated. The re-isolated fungi were identified and confirmed based on the morphological characteristics and disease symptoms exhibited by the leaves with the original fungi. Statistical analyses were performed by one-way analysis of variance (ANOVA) using SPSS Statistics 22 software (IBM Corp., Armonk, NY, USA).

Sample Collection and Fungal Isolation
A total of 428 soil samples were collected from seven sampling sites (A to G) in four regions in GuangXi ( Figure 1, Table 1). The fungi with branched conidiophores producing cylindrical conidia and with stipe extensions terminating in a vesicle with a characteristic shape were grouped as Calonectria. A total of 188 soil samples, which accounted for 43.9% of all sampled soil samples, were positive for Calonectria isolates with branched conidiophores, cylindrical macroconidia, and sphaeropedunculate or clavate vesicles. For each sample, a single conidium culture was isolated from white masses of conidiophores with typical morphological characteristics of Calonectria species. In total, 188 Calonectria isolates were obtained from 188 soil samples. The percentage of soil samples that yielded Calonectria ranged from 5.0% to 62.3% at the seven sampling sites (Table 1).

Sequencing
DNA extraction and sequence comparisons of all 188 Calonectria isolates were performed ( Table 2). The tef1, tub2, cmdA, and his3 gene regions of all 188 isolates were amplified. The obtained sequence fragments for the tef1, tub2, cmdA, and his3 gene regions were approximately 520, 600, 690, and 460 bp, respectively. Based on the sequences of the tef1, tub2, cmdA, and his3 loci, the genotypes of all 188 sequenced isolates were determined. A total of 32 genotypes were identified ( Table 2).

Phylogenetic Analyses
For the 188 isolates sequenced in this study, one to two isolates of each genotype determined by tef1, tub2, cmdA, and his3 sequences were selected for phylogenetic analyses. A total of 47 representative isolates representing 32 genotypes were selected ( Table 2). The sequences of 69 isolates presenting 40 published Calonectria species closely related to the Calonectria isolates obtained in the present study were downloaded from GenBank and used for phylogenetic analyses based on four individual gene regions and the combination of those regions (Table 3).
For BI phylogenetic analyses of each dataset, GTR+I, TPM2uf+I+G, TIM1+G, TPM2uf+I+G, and GTR+I+G models were selected for tef1, tub2, cmdA, his3, and the combination of those regions, respectively. The overall topologies generated from the ML analyses and the BI analyses for each dataset were similar. The ML tree with bootstrap support values and the posterior probabilities obtained from BI are presented in Figure 2 and Supplementary Figures S1-S4.
The 47 Calonectria isolates were divided into five groups (Groups A to E) based on tef1, tub2, cmdA, his3, and combined tef1/tub2/cmdA/his3 analyses (Figure 2 and Supplementary Figures S1-S4). The phylogenetic analyses showed that the isolates in Groups A, B, C, and D belong to the C. kyotensis species complex, while the isolates in Group E belong to the C. reteaudii species complex.
The isolates in Group A represented 19 genotypes based on the sequences of four gene regions ( Table 2). The phylogenetic analyses showed that these isolates were grouped with Calonectria aconidialis L. Lombard, Crous and S.F. Chen based on the tef1, cmdA, and his3 trees (Supplementary Figures S1, S3 and S4). In the tub2 tree, the isolates were clustered directly with or most closely to C. aconidialis, Calonectria asiatica Crous and Hywel-Jones, and Calonectria uniseptate Gerlach (Supplementary Figure S2), and were grouped with C. aconidialis according to the combined tef1/tub2/cmdA/his3 tree ( Figure 2). Therefore, the isolates in Group A were identified as C. aconidialis. The isolates in Group B represented one genotype (Table 2). These isolates were clustered with Calonectria kyotensis Terash. in the tef1, tub2, and his3 trees (Supplementary Figures S1, S2, and S4), and were clustered directly with or most closely to C. kyotensis and C. uniseptate in the cmdA tree (Supplementary Figure  S3). According to the combined tef1/tub2/cmdA/his3 tree, these isolates were grouped with C. kyotensis (Figure 2), and therefore isolates in Group B were identified as C. kyotensis. The isolates in Group C represented 10 genotypes (Table 2) and were clustered with Calonectria hongkongensis Crous in the tef1, tub2, cmdA, and his3 trees and the four-gene combined phylogenetic tree (Figure 2 and Supplementary Figures S1-S4). The isolates in Group C were identified as C. hongkongensis. The isolate in Group D represented one genotype ( Table 2). This isolate was clustered with Calonectria chinensis (Crous) L. Lombard, M.J. Wingf. and Crous in the cmdA and his3 trees ( Supplementary Figures S3 and S4). The isolate was clustered directly with or most closely to C. chinensis in the tef1 and tub2 trees ( Supplementary Figures S1 and S2). The isolate was clustered with C. chinensis based on the combined tef1/tub2/cmdA/his3 tree ( Figure 2). Consequently, the isolate was identified as C. chinensis.
The isolates in Group E represented one genotype (Table 2). These isolates were clustered with C. pseudoreteaudii in the tef1, tub2, and his3 trees (Supplementary Figures S1,  S2 and S4). These isolates were grouped with C. pseudoreteaudii and Calonectria reteaudii (Bugnic.) C. Booth in the cmdA tree (Supplementary Figure S3). According to the combined tef1/tub2/cmdA/his3 tree, these isolates were grouped with C. pseudoreteaudii (Figure 2). Therefore, the isolates in Group E were identified as C. pseudoreteaudii.

Pathogenicity Tests
Forty isolates representing the five Calonectria species, C. aconidialis (21 isolates), C. hongkongensis (12 isolates), C. kyotensis (three isolates), C. pseudoreteaudii (three isolates), and C. chinensis (one isolate), were used for pathogenicity tests on the leaves of three Eucalyptus genotypes ( Table 2, Figures 4 and 5). All 40 isolates and the positive control produced disease spots and lesions on the leaves of the inoculated seedlings. No disease symptoms were observed in the leaves of the negative control seedlings (Figures 4 and 5). Calonectria species with the same morphological characteristics as the originally inoculated fungi were successfully re-isolated from the diseased tissues of the inoculated leaves. No Calonectria isolates were re-isolated from the leaves of the negative control seedlings. Thus, Koch's postulates were fulfilled. Two pathogenicity tests were performed, and ANOVA showed that the two pathogenicity tests were significantly different (p < 0.05). Consequently, the data from each experiment were analyzed separately.

Pathogenicity Tests
Forty isolates representing the five Calonectria species, C. aconidialis (21 isolates), C. hongkongensis (12 isolates), C. kyotensis (three isolates), C. pseudoreteaudii (three isolates), and C. chinensis (one isolate), were used for pathogenicity tests on the leaves of three Eucalyptus genotypes ( Table 2, Figures 4 and 5). All 40 isolates and the positive control produced disease spots and lesions on the leaves of the inoculated seedlings. No disease symptoms were observed in the leaves of the negative control seedlings (Figures 4 and 5). Calonectria species with the same morphological characteristics as the originally inoculated fungi were successfully re-isolated from the diseased tissues of the inoculated leaves. No Calonectria isolates were re-isolated from the leaves of the negative control seedlings. Thus, Koch's postulates were fulfilled. Two pathogenicity tests were performed, and ANOVA showed that the two pathogenicity tests were significantly different (p < 0.05). Consequently, the data from each experiment were analyzed separately.

Discussion
In this study, 428 soil samples were collected from seven Eucalyptus plantations in multiple regions of GuangXi in southern China. Based on their morphological characteristics, 188 Calonectria isolates were obtained. Of these, 188 isolates were identified based The results of the pathogenicity tests showed that some isolates of C. aconidialis, C. hongkongensis, and C. kyotensis generated significantly longer lesions than the positive control on each of the three Eucalyptus genotypes in both experiments (p < 0.05). For exam-ple, C. aconidialis isolates (CSF16507, CSF16520, CSF16557, CSF16582, CSF16648, CSF16693, CSF16706, CSF17110, CSF17130, and CSF17142), C. hongkongensis isolate CSF17125, and C. kyotensis isolate CSF16776 produced significantly longer lesions than positive control isolate CSF13317 (C. pseudoreteaudii) on the three Eucalyptus genotypes in both experiments (Figures 4 and 5).
The resistance of different Eucalyptus genotypes to Calonectria isolates within the same species was inconsistent. For example, Eucalyptus genotypes CEPT1900 and CEPT1901 were significantly more tolerant than genotype CEPT1902 in both experiments (p < 0.05) to C. aconidialis isolates (CSF16470, CSF16522, CSF16527, CSF16742, and CSF17130), and C. hongkongensis isolate CSF17118 was significantly more tolerant than genotype CEPT1902 in both experiments (p < 0.05). Eucalyptus genotype CEPT1901 was significantly more tolerant to C. aconidialis isolate CSF16599 than to the other two Eucalyptus genotypes in both experiments (p < 0.05) (Figures 4 and 5).

Discussion
In this study, 428 soil samples were collected from seven Eucalyptus plantations in multiple regions of GuangXi in southern China. Based on their morphological characteristics, 188 Calonectria isolates were obtained. Of these, 188 isolates were identified based on multigene phylogenetic inferences. These isolates were identified as C. aconidialis, C. hongkongensis, C. pseudoreteaudii, C. kyotensis, and C. chinensis. Pathogenicity tests indicated that all five Calonectria species were pathogenic among the three tested Eucalyptus genotypes.
This study showed that Calonectria fungi in the C. kyotensis species complex were widely distributed in the soil of Eucalyptus plantations in southern China. Calonectria fungi were isolated from 43.9% of the soil samples. Except for C. pseudoreteaudii, which reside in the C. reteaudii species complex, the other four species resided in the C. kyotensis species complex.
The four species in the C. kyotensis species complex accounted for 97.9% of all the isolates obtained in this study. This is consistent with the results of previous studies showing that Calonectria species in the C. kyotensis species complex, especially C. aconidialis, C. hongkongensis, and C. kyotensis, are the dominant species distributed in the soil of Eucalyptus plantations in southern China [9,[23][24][25]. In addition to soil isolation, C. aconidialis, C. kyotensis, C. hongkongensis, and C. chinensis were also occasionally isolated from diseased Eucalyptus tissues [8,28,55]. Calonectria hongkongensis also caused fruit rot in rambutan (Nephelium lappaceum L.) in Puerto Rico [57]. The Calonectria species in the C. kyotensis species complex isolated from the soil in this study can cause disease in Eucalyptus trees.
In this study, one species in the C. reteaudii species complex, C. pseudoreteaudii, was isolated from the soil of three Eucalyptus plantation sites. This fungus has been extensively isolated from diseased Eucalyptus tissues (leaves and branches) in plantations in FuJian, GuangXi, GuangDong, and HaiNan Provinces in southern China [5,[7][8][9]21,43,58]. Calonectria pseudoreteaudii is considered one of the key causal agents of Eucalyptus leaf blight in southern China. Except for Eucalyptus trees, C. pseudoreteaudii caused leaf spots in Macadamia F. Muell. sp. in China and Laos [59,60] and caused leaf spot and stem blight in Vaccinium corymbosum L. in China [61]. The results of this and previous studies suggested that C. pseudoreteaudii is an important pathogen in many plant species with a wide geographic distribution.
The inoculation results indicated that isolates of C. aconidialis, C. hongkongensis, and C. kyotensis produced significantly longer lesions than those of the positive control on the three Eucalyptus genotypes. These results highlighted that Calonectria species dominantly distributed in the soil were potential threats to Eucalyptus plantations in southern China.
One of the most effective measures to control Eucalyptus leaf blight caused by Calonectria species is selecting disease-resistant Eucalyptus genotypes. Eucalyptus genotypes resistant to Eucalyptus leaf blight have been selected in Australia, Brazil, China, India, and South Africa [62][63][64][65][66][67].
The pathogenicity tests in this study indicated that some Calonectria isolates within the same species were significantly different in their virulence from the tested Eucalyptus genotypes. The resistance of different Eucalyptus genotypes to Calonectria isolates within the same species was inconsistent. This is consistent with the results of previous studies [9,29]. Variations in plant pathogen intra-species pathogenicity and differences in plant pathogen resistance are common in some pathogens and plants [68][69][70]. This is the result of the evolution of both pathogenicity and virulence of plant pathogens, pathogen and plant genetic regulation, plant pathogen co-evolution, and other factors [71][72][73]. The results of this and previous studies suggested that in the process of selecting disease-resistant Eucalyptus genotypes, many isolates of each Calonectria species with different pathogenicities and many Eucalyptus genotypes should be tested.

Supplementary Materials:
The following supporting information can be downloaded at: https://www. mdpi.com/article/10.3390/jof9080802/s1, Figure S1: Phylogenetic tree of Calonectria species based on maximum likelihood (ML) analyses of the tef1 gene sequences in this study; Figure S2: Phylogenetic tree of Calonectria species based on maximum likelihood (ML) analyses of the tub2 gene sequences in this study; Figure S3: Phylogenetic tree of Calonectria species based on maximum likelihood (ML) analyses of the cmdA gene sequences in this study; Figure S4: Phylogenetic tree of Calonectria species based on maximum likelihood (ML) analyses of the his3 gene sequences in this study.
Author Contributions: S.C. conceived and designed the experiments. S.C. collected the samples. W.W. collected the samples and performed laboratory work, pathogenicity tests, and data analysis. All authors analyzed and checked the data. All authors wrote and revised the paper. All authors agreed to be accountable for all aspects of the work. All authors have read and agreed to the published version of the manuscript.