Wide Distribution of Teratosphaeria epicoccoides and T. destructans Associated with Diseased Eucalyptus Leaves in Plantations in Southern China

Species of Mycosphaerellaceae and Teratosphaeriaceae represent over 40% of the fungi identified on eucalypt leaves worldwide. These include some important pathogens that mainly cause leaf blight and spot, and result in increasingly negative impacts on global commercial eucalypt industries. Eucalyptus plantations are commonly cultivated in southern China for solid wood and pulp products. However, the species diversity and geographic distribution of Mycosphaerellaceae and Teratosphaeriaceae, associated with diseased plantation Eucalyptus leaves in China, have not been clarified. In this study, we conducted the first systematic surveys and sample collections of Mycosphaerellaceae- and Teratosphaeriaceae-like fungi from diseased plantation Eucalyptus leaves in southern China. In total, 558 isolates were obtained from 59 sampled sites in five provinces. One isolate was isolated from each tree. According to the disease symptoms, conidia morphological characteristics, and DNA sequence comparisons of ITS, tef1 and tub2 gene regions. The 558 isolates were identified as Teratosphaeria epicoccoides (312 isolates; 55.9%) and T. destructans (246 isolates, 44.1%). Both species were widely distributed in the sampled regions in southern China. The genotypes of T. epicoccoides and T. destructans were determined based on ITS, tef1, and tub2 sequences. The results showed that multiple genotypes of each species of T. epicoccoides and T. destructans exist in China. Additionally, isolates with multiple genotypes were obtained in all five sampled provinces. These results suggest that both T. epicoccoides and T. destructans are not clonal. This study proved that both T. epicoccoides and T. destructans are dominant species and widely distributed on diseased Eucalyptus leaves in southern China. The wide geographic distribution and potential high genetic diversity pose challenges for the disease management of Teratosphaeria leaf blight and leaf spot in China.


Introduction
In China, eucalypts plantations have expanded rapidly to meet the increasing demand for wood and pulp [1].Occupying approximately 5.4 million hm 2 in China in 2018, the plantations account for 6.8% of the national plantation area and provide more than one-third of the total national commercial timber production [2].Eucalyptus plantations are mainly planted in the southern regions of China, especially in Guangxi, Guangdong, Yunnan, and Fujian Provinces.Mostly, selected genotypes of Eucalyptus urophylla × E. grandis hybrids are planted and grown [3].
Globally, leaf diseases on Eucalyptus caused by the fungi of Mycosphaerellaceae and Teratosphaeriaceae are widely distributed [20][21][22][23].In recent years, a number of novel species of Mycosphaerellaceae and Teratosphaeriaceae have been isolated from diseased Eucalyptus leaves and described [23][24][25], some of which are known to cause leaf diseases on Eucalyptus.
According to the research findings of Crous et al. [23], species belonging to Mycosphaerellaceae and Teratosphaeriaceae demonstrate a high species diversity on a global scale.Species from these two families represent over 40% of the fungi identified on eucalypts leaves worldwide [23].
In China, several species of Mycosphaerellaceae and Teratosphaeriaceae have been implicated as a cause of Eucalyptus leaf spot/blight [26][27][28][29][30].Of the foliar pathogens belonging to Mycosphaerellaceae and Teratosphaeriaceae isolated from Eucalyptus leaves in China, only T. destructans (causing Eucalyptus leaf blight) [26] and Pseudocercospora chiangmaiensis (responsible for Eucalyptus circular leaf spot) [28,31] were identified based on both DNA sequence comparisons and morphological characteristics.The identification of the remaining species causing leaf spot/blight relied solely on morphological features [32][33][34][35][36][37].More than 600 Eucalyptus species are native to Australia, with only a few species endemic to Papua New Guinea, some parts of Indonesia and the Philippines [38,39].Many fungi of Mycosphaerellaceae and Teratosphaeriaceae have been described from Eucalyptus foliage in Australia, and most were considered to be endemic [23,40,41].Previous studies have shown that Mycosphaerellaceae and Teratosphaeriaceae exhibit high species diversity; species in these two families are the predominant fungi on diseased Eucalyptus leaves globally [23].In China, research on the species diversity of Mycosphaerellaceae and Teratosphaeriaceae fungi on diseased Eucalyptus leaves is very limited, and the identification of some of these species is not accurate because of the lack of DNA sequence data [26,27,29,30].Furthermore, there are limited systematic studies on their geographical distribution.Recently, we collected diseased Eucalyptus leaves with typical mature fruiting structures of Mycosphaerellaceae and Teratosphaeriaceae from Eucalyptus plantations in Yunnan, Guangxi, Hainan, Guangdong, and Fujian Provinces in southern China.We subsequently isolated these fungi.The aims of this study were to: (i) identify these fungi based on DNA sequence comparisons of multi-gene regions and morphological characteristics; and (ii) explore the geographical distribution characteristics of Mycosphaerellaceae and Teratosphaeriaceae species on diseased Eucalyptus leaves in southern China.

Disease Symptoms, Samples, and Fungal Isolations
The study was mainly conducted from July to October 2022, and from February to April 2023.The sampled regions had high temperatures (20 • C-35 • C) and high levels of humidity (60-90%).Diseases caused by Mycosphaerellaceae and Teratosphaeriaceae were surveyed on Eucalyptus plantations in the Guangxi, Guangdong, Yunnan, Hainan, and Fujian provinces in southern China.At most sampling sites, the trees were 1 to 2 years old.E. urophylla × E. grandis hybrids were dominant, although a few genotypes of E. urophylla, E. urophylla × E. tereticomis, and E. urophylla × E. pellita were also surveyed (Table 1).In the plantations, the leaves of trees infected by Mycosphaerellaceae and Teratosphaeriaceae fungi, resulting in leaf spot, vein delimitation, chlorosis, and intense defoliation were identified (Figure 1A,B).In some surveyed regions, all trees in the plantations were infected (Figure 1A,B).Based on the diseased symptoms, two groups of fungi causing different diseases were observed.The first group of fungi mainly infected mature and old leaves and produced abundant small spots on the infected leaves.The second group of fungi mainly infected the juvenile leaves, but affected some mature leaves as well, and resulted in water-soaked, chlorosis, and wrinkled symptoms on the infected leaves (Figure 1C-N).Disease symptoms of the first group of fungi were frequently observed in most surveyed Eucalyptus plantations.Disease symptoms with the second group of fungi were observed occasionally in some regions in southern China (Figure 1O-R).Diseased leaves with typical fruiting structures of Mycosphaerellaceae and Teratosphaeriaceae were collected from 3-59 trees, or approximately 30 trees, at the majority of sampling sites, depending on the area of the sampled plantation.Diseased leaf samples were transported to the laboratory for morphological examination, isolation, and further assessments.
Fungal isolates in the conidiomata with the typical morphological characteristics of Mycosphaerellaceae and Teratosphaeriaceae [23] were isolated from diseased leaves.The conidia masses in the conidiomata were scattered onto 2% malt extract agar (MEA) (20 g malt extract powder and 20 g agar powder per liter of water; malt extract powder was obtained from the Beijing Shuangxuan Microbial Culture Medium Products factory, Beijing, China; the agar powder was obtained from Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) with sterile needles under a stereoscopic microscope (AxioCam Stemi 2000C, Carl Zeiss, Jena, Germany).After incubation at 25 • C for 6-10 h, the germinated conidia were transferred individually onto fresh 2% MEA under the dissection microscope and incubated at 25 • C for four weeks to obtain single-conidium cultures.One single-conidium culture was obtained from leaves of each sampled tree.All the single-conidium cultures were deposited in the culture collection (CSF) of the Research Institute of Fast-growing Trees (RIFT), Chinese Academy of Forestry (CAF), in Zhanjiang, Guangdong Province, China.

DNA Extraction, PCR Amplification, and Sequencing
All isolates obtained were used for DNA extraction and sequence analyses.DNA was extracted from four-week-old cultures.Mycelia were scraped using a sterilized scalpel and transferred to 2.0 mL Eppendorf tubes.The total genomic DNA was extracted using the CTAB protocol, as described by van Burik et al. [42].The extracted DNA was dissolved in 30 µL of TE buffer (1 M Tris-HCl and 0.5 M EDTA, pH = 8.0).To degrade the RNA, 2.5 µL RNase (10 mg/mL) was added at 37 • C for one hour.The DNA concentration was measured using a NanoDrop 2000 spectrometer (Thermo Fisher Scientific, Waltham, MA, USA).
All the PCR products of all isolates obtained in this study were sequenced in both the forward and reverse directions with the same primers used in the PCR amplification.Sequence reactions were conducted at the Beijing Genomics Institute, Guangzhou, China.All sequences obtained were edited using MEGA v. 7.0 software [49], and were deposited in GenBank (https://www.ncbi.nlm.nih.gov,accessed on 6 December 2023).The ITS, tef1, and tub2 gene regions were sequenced for all isolates obtained in this study.

Multi-Gene Phylogenetic Analyses and Species Identification
All isolates obtained in this study were genotyped by their ITS, tef1, and tub2 sequences.Based on the genotypes generated by the ITS, tef1, and tub2 sequences, sequences of two isolates for each ITS-tef1-tub2 genotype were selected for phylogenetic analyses.Isolates with the same genotype were considered to be the same species.
The preliminary identities of the isolates obtained in this study were determined by conducting a standard nucleotide BLAST search using all the generated sequences of ITS, tef1, and tub2.The BLAST results indicated that the isolates obtained in this study were grouped in the genus Teratosphaeria.The sequences of the type strains closely related to the Teratosphaeria isolates sequenced in the current study were downloaded from the NCBI database (http://www.ncbi.nlm.nih.gov/accessed on 15 October 2023) and used for phylogenetic analyses (Table 3).Sequences downloaded from the NCBI database and sequences generated in this study were aligned using MAFFT online v. 7 (http://mafft.cbrc.jp/alignment/server/(accessed on 2 November 2023)) [50], with the iterative refinement method (FFT-NS-i setting).The alignments were further edited manually with MEGA v. 7.0 software [49] when necessary.Sequences of each of the ITS, tef1, and tub2 gene regions, as well as the combination of these three gene regions, were analyzed.
Maximum likelihood (ML) analyses were conducted for the three individual gene sequences of ITS, tef1, and tub2, as well as for a concatenated dataset of all three genes.ML analyses were performed with RaxML v. 8.2.4 on the CIPRES Science Gateway v. 3.3 [51], with default GTR substitution matrix and 1000 bootstrap replicates [52].Phylogenetic trees were viewed using MEGA v. 7.0 [49].Sequence data of one isolate of Staninwardia suttonii (CBS 120061) were used as the outgroup [30].

Fungal Isolations
Diseased leaves with conidiomata of Mycosphaerellaceae and Teratosphaeriaceae were collected from 59 sites in five provinces in southern China (Table 1; Figure 2).Single conidia from the conidiomata on the diseased leaves were transferred to fresh 2% MEA for fungal isolation.These conidia exhibited the typical morphological characteristics of Teratosphaeria species.Two groups of fungi corresponding to the disease symptoms were observed.The conidia of the first group of fungi were generally brown, and straight to slightly curved in shape.The number of septa in the conidia ranged from one to seven; the majority of conidia had three to five septa (Figure 3).The conidia of the second group of fungi were light brown, variously curved, and rarely straight.The conidia had one to three septa; the majority of conidia had three septa (Figure 4).The conidia of both groups of fungi were base truncate and apex obtuse.The conidia of the first group of fungi were wider, straighter, and darker than those of the second group (Figures 3 and 4).These two groups of fungi had the typical morphological characteristics of T. epicoccoides and T. destructans, respectively [43,53].In total, 558 isolates were obtained from the 59 sites.Three to twenty isolates were obtained from each sampled site, depending on the number of samples collected and the morphological variations in the conidia on the diseased leaves (Table 1).

Fungal Isolations
Diseased leaves with conidiomata of Mycosphaerellaceae and Teratosphaeriaceae were collected from 59 sites in five provinces in southern China (Table 1; Figure 2).Single conidia from the conidiomata on the diseased leaves were transferred to fresh 2% MEA for fungal isolation.These conidia exhibited the typical morphological characteristics of Teratosphaeria species.Two groups of fungi corresponding to the disease symptoms were observed.The conidia of the first group of fungi were generally brown, and straight to slightly curved in shape.The number of septa in the conidia ranged from one to seven; the majority of conidia had three to five septa (Figure 3).The conidia of the second group of fungi were light brown, variously curved, and rarely straight.The conidia had one to three septa; the majority of conidia had three septa (Figure 4).The conidia of both groups of fungi were base truncate and apex obtuse.The conidia of the first group of fungi were wider, straighter, and darker than those of the second group (Figures 3 and 4).These two groups of fungi had the typical morphological characteristics of T. epicoccoides and T. destructans, respectively [43,53].In total, 558 isolates were obtained from the 59 sites.Three to twenty isolates were obtained from each sampled site, depending on the number of samples collected and the morphological variations in the conidia on the diseased leaves (Table 1).Both groups of fungi were isolated from plantations of E. urophylla, E. urophylla × E. grandis, E. urophylla × E. pellita and E. urophylla × E. tereticomis, and most especially from Both groups of fungi were isolated from plantations of E. urophylla, E. urophylla × E. grandis, E. urophylla × E. pellita and E. urophylla × E. tereticomis, and most especially from multiple genotypes of E. urophylla × E. grandis, which were widely planted in the sampled regions.In addition, the first group of fungi were also frequently observed on the majority of other Eucalyptus genotypes in the plantations, including species of E. camaldulensis, E. grandis, E. pellita and their hybrids with E. urophylla.T. destructans is limited to certain species of E. grandis, E. urophylla, E. tereticornis and their hybrids.

Multi-Gene Phylogenetic Analyses and Species Identification
The BLAST results indicated that the 558 isolates obtained and sequenced in this study were mainly categorized into two groups.These two groups of fungi were similar to T. epicoccoides (Group A) and T. destructans (Group B), respectively.Amplicons generated for the ITS, tef1, and tub2 gene regions of fungi similar to T. epicoccoides were approximately 500, 350, and 350 bp, respectively.The sequences of ITS, tef1, and tub2 gene regions of fungi similar to T. destructans were approximately 290, 250, and 350 bp, respectively.Sequences of two isolates for each genotype generated by the three genes were used in the analyses.Sequences of ex-type specimen strains and other strains of 29 Teratosphaeria species, including T. epicoccoides and T. destructans, closely related to isolates obtained in the current study, were downloaded from GenBank for sequence comparisons and phylogenetic analyses (Table 3).
Based on the phylogenetic analyses of the ITS, tef1, tub2, and the combined datasets, the isolates of two groups of fungi, Group A and Group B in this study, were most closely related to T. epicoccoides and T. destructans, respectively.The isolates of Group A were consistently grouped with, or close to, the ex-type isolate CMW 5348 of T. epicoccoides in each of the ITS, tef1, tub2, and the combined dataset trees (Figures 5-8).Additionally, some isolates of Group A formed independent clades in each of the four phylogenetic trees (Figures 5-8).This is consistent with the phylogenetic analysis results presented by Taole et al. [43].Analysis of the sequence data of the three regions resulted in incongruent genealogies.There was no evidence of distinct species boundaries.Isolates of Group A were identified as T. epicoccoides.
In Group B, all isolates were grouped with ex-type isolate CBS 111370 of T. destructans in the tub2 tree (Figure 8).Additionally, some isolates of Group B formed independent clades in each of the ITS and tef1 phylogenetic trees.However, these isolates did not form consistently independent clades, nor were they supported by high bootstrap values in both the ITS and tef1 phylogenetic trees (Figures 6 and 7).For example, isolates of genotypes CAA and CBA were grouped together and formed one independent clade with high bootstrap value (89%) in the ITS tree (Figure 6).These isolates were grouped with ex-type isolates CBS 111369 and CBS 111370 of T. destructans in the tef1 tree (Figure 7).The results suggest that these reflect intraspecific sequence differences rather than interspecies variation.Combining the phylogenetic analysis results of ITS, tef1, tub2 and the datasets (Figures 5-8), the isolates in Group B were identified as T. destructans.multiple genotypes of E. urophylla × E. grandis, which were widely planted in the sampled regions.In addition, the first group of fungi were also frequently observed on the majority of other Eucalyptus genotypes in the plantations, including species of E. camaldulensis, E. grandis, E. pellita and their hybrids with E. urophylla.T. destructans is limited to certain species of E. grandis, E. urophylla, E. tereticornis and their hybrids.

Multi-Gene Phylogenetic Analyses and Species Identification
The BLAST results indicated that the 558 isolates obtained and sequenced in this study were mainly categorized into two groups.These two groups of fungi were similar to T. epicoccoides (Group A) and T. destructans (Group B), respectively.Amplicons generated for the ITS, tef1, and tub2 gene regions of fungi similar to T. epicoccoides were approximately 500, 350, and 350 bp, respectively.The sequences of ITS, tef1, and tub2 gene regions of fungi similar to T. destructans were approximately 290, 250, and 350 bp, respectively.Sequences of two isolates for each genotype generated by the three genes were used in the analyses.Sequences of ex-type specimen strains and other strains of 29 Teratosphaeria species, including T. epicoccoides and T. destructans, closely related to isolates obtained in the current study, were downloaded from GenBank for sequence comparisons and phyloge- isolates of Group A formed independent clades in each of the four phylogenetic trees (Figures [5][6][7][8].This is consistent with the phylogenetic analysis results presented by Taole et al. [43].Analysis of the sequence data of the three regions resulted in incongruent genealogies.There was no evidence of distinct species boundaries.Isolates of Group A were identified as T. epicoccoides.In Group B, all isolates were grouped with ex-type isolate CBS 111370 of T. destructans in the tub2 tree (Figure 8).Additionally, some isolates of Group B formed independent clades in each of the ITS and tef1 phylogenetic trees.However, these isolates did not form consistently independent clades, nor were they supported by high bootstrap values in both the ITS and tef1 phylogenetic trees (Figures 6 and 7).For example, isolates of genotypes CAA and CBA were grouped together and formed one independent clade with high bootstrap value (89%) in the ITS tree (Figure 6).These isolates were grouped with ex-type isolates CBS 111369 and CBS 111370 of T. destructans in the tef1 tree (Figure 7).The results

Distribution of Teratosphaeria Species
Based on the DNA sequence comparisons of ITS, tef1, and tub2 sequences, the 558 isolates obtained from 59 sampling sites in this study were identified as T. epicoccoides (312 isolates; 55.9%) and T. destructans (246 isolates, 44.1%).T. epicoccoides was isolated and identified in 56 (accounting 95%) sampling sites, and T. destructans was obtained from 27 (accounting 46%) sampling sites.These two species were all obtained from 24 (accounting for 41%) sampling sites (Figure 2).Determined by ITS, tef1, and tub2 sequences, 21 genotypes were generated for the 291 T. epicoccoides isolates (the genotypes of 21 isolates were not clear because the sequences of three gene regions were not all obtained) (Table 4).Six genotypes were generated for the 242 T. destructans isolates (the genotypes of four isolates were not clear because the sequences of three gene regions were not all obtained) (Table 5).The ratios of the genotype number to the isolate number of T. epicoccoides and T. destructans were all highest in Hainan Province.

Discussion
In this study, systematical disease surveys were conducted to collect diseased Eucalyptus leaves with typical fruiting structures of Mycosphaerellaceae and Teratosphaeriaceae in the core Eucalyptus plantation regions in China.In total, 558 isolates were identified according to disease symptoms and morphological characteristics and were mainly based on DNA sequence comparisons of three gene regions.These fungi were identified as T. epicoccoides and T. destructans.The results of this study indicate that T. epicoccoides and T. destructans are widely distributed in the core Eucalyptus planting regions in southern China.
This study has clarified and expanded the geographic distribution of Teratosphaeria species associated with diseased Eucalyptus leaves in southern China.Teratosphaeria epicoccoides was initially described in 1992.It was indicated that this species is distributed in many countries and regions worldwide.The leaf specimens used for the initial description included a total of 25 species of Eucalyptus and Corymbia in Australia, Brazil, Argentina, Zambia, India, and Ethiopia [54].Currently, T. epicoccoides is widely reported on Eucalyptus leaves globally [41,43].Teratosphaeria destructans was initially reported and described in 1996 on the leaves of E. grandis in Indonesia, along with some other unknown Eucalyptus species [53].Currently, T. destructans is reported in a global range, including countries in Africa such as South Africa, and countries in Asia such as China, Thailand, and East Timor [41,55].In China, T. epicoccoides and T. destructans were both first isolated from leaves of E. urophylla in Guangdong Province in 2006 [13].Prior to this study, the distribution reports of these two species in China were both limited to Guangdong Province [13,26].The results of this study suggest that T. epicoccoides is distributed in areas that are majorityplanted with Eucalyptus in southern China, and T. destructans is also widely distributed throughout large regions in this country.
DNA sequence comparisons were considered a reliable method for the classification and identification of Mycosphaerellaceae and Teratosphaeriaceae [23,30].For species of Mycosphaerellaceae and Teratosphaeriaceae, traditional classification has primarily relied on morphological characteristics.When classifying based on morphological characteristics, the identification of species of Mycosphaerellaceae and Teratosphaeriaceae is influenced by the conserved morphological features of their respective sexual morphs [56][57][58].Scientists have therefore shifted the focus of classifying and identifying these species mostly towards their asexual morphs [59][60][61].However, similar asexual morphologies have also independently evolved from different taxa, adding further complexity to the taxonomy of these species [62].In 2014, Quaedvlieg et al. proposed that the accurate classification of genera and species within Mycosphaerellaceae and Teratosphaeriaceae cannot be achieved solely through morphological comparisons.Instead, it requires the integration of molecular data [30].The research results indicated that the ITS gene serves as a primary barcode locus to distinguish taxa in Mycosphaerella and Teratosphaeria associated with Eucalyptus leaf disease.The ITS gene is easily generated and has the most extensive dataset available; therefore, tef1, tub, or rpb2 represent useful secondary barcode loci [30].In this study, species identification was primarily based on the comparison of three gene sequences of ITS, tef1, and tub2.These three gene sequences have been widely employed to differentiate both intra-and inter-specific variations among species in Teratosphaeriaceae [25,43,55].
To date, eight species of Mycosphaerellaceae and Teratosphaeriaceae isolated from Eucalyptus leaves in China have been identified based on DNA sequence comparisons.These species include Neoceratosperma yunnanensis, Pallidocercospora crystallina, Paramycosphaerella marksii, Pseudocercospora flavomarginata, Pse.gracilis, Pse.haiweiensis, Teratosphaeria destructans, and T. epicoccoides [26,27,29,30,63].With the exception of two species of Teratosphaeria, the other six species reside in Mycosphaerellaceae.It is still unknown whether these eight species are all pathogenic to Eucalyptus in China because no pathogenicity tests have been conducted.
One shortcoming of this study is that the pathogenicity of the two identified Teratosphaeria species was not investigated.At present, on a global scale, methods for assessing the pathogenicity of Mycosphaerellaceae and Teratosphaeriaceae species isolated from diseased Eucalyptus leaves include spraying ascospore suspensions, conidial suspensions, hyphal fragment suspensions, or a mixture of spores and hyphal fragment suspensions onto Eucalyptus seedlings in controlled environments.Additional inoculation methods involve applying ascospore masses or lesions from diseased leaf areas onto Eucalyptus seedling leaves [44,57,64].Conidial suspensions are considered to be a typical method for evaluating the pathogenicity of Mycosphaerellaceae and Teratosphaeriaceae species; however, this is not possible for species that do not sporulate sufficiently in culture.Very limited research has been conducted on the pathogenicity of T. destructans and T. epicoccoides on Eucalyptus.The Teratosphaeria isolates obtained in the current study failed to produce conidia on media.In order to test the pathogenicity of T. destructans and T. epicoccoides, it is vital to find a way to sufficiently induce the sporulation of these fungi in culture or find a new method to test their pathogenicity.For example, the hyphal fragment suspensions were replaced with conidial suspensions to test the pathogenicity of Calonectria pseudoreteaudii [65].Hyphal fragment suspension may also be used to assess the pathogenicity of Teratosphaeria species, although this need to be evaluated.Previous research results indicated that T. epicoccoides should be indigenous to eastern Australia, and spread to western Australia and other regions of the world [41,66].T. destructans is recognized as absent from Australia.The origin of T. destructans remains unknown, but it is likely to have originated in Indonesia or East Timor [41,[67][68][69].Research results support the hypothesis that T. epicoccoides and T. destructans are spread via the human-mediated movement of infected plants and seeds [41,66,70].This suggests the need for much more stringent and more sophisticated biosecurity measures, such as the use of metabarcoding approaches to test seedlots and plant materials under quarantine [41,67,68].
The results of this study confirmed that both T. epicoccoides and T. destructans are dominant species on diseased Eucalyptus leaves in China.Both species are widely distributed in Eucalyptus plantations that are mainly distributed across regions of southern China.Although previous studies have indicated that T. epicoccoides and T. destructans are widely distributed on Eucalyptus leaves globally [41,44], research on these species on diseased Eucalyptus leaves specifically in China is limited.During the sampling process in the current study, we found that the severity caused by each species of T. epicoccoides and T. destructans on different genotypes of E. urophylla × E. grandis were different.Research results showed that significant differences in resistance exist among the six tested E. grandis × E. urophylla genotypes to the inoculated T. destructans [71].It is suggested that breeding and selection of Eucalyptus tolerant/resistant species, hybrids and clones are needed for the future management of leaf diseases caused by Teratosphaeria species [41].Future research should focus on exploring pathogenicity testing methods for T. epicoccoides and T. destructans and clarifying their pathogenic characteristics.This will guide the selection of disease-resistant Eucalyptus genotypes for these two widely distributed species.

Figure 1 .
Figure 1.Disease symptoms associated with Teratosphaeria epicoccoides and T. destructans on Eucalyptus plantations in southern China: (A,B) trees in 2-year-old Eucalyptus urophylla × E. grandis plantations associated with T. epicoccoides and T. destructans.Leaves of the whole trees were infected and resulted in intense defoliation in the tree growth season; (C,D) typical leaf spot, vein delimitation, and chlorosis symptoms on leaves of E. urophylla × E. grandis associated with T. epicoccoides.New leaves on the top of shoots emerged after infection (C); (E,F) all leaves of 0.5-year-old E. urophylla × E. grandis trees in one plantation infected by T. destructans; (G,H) typical disease symptoms on E. urophylla × E. grandis leaves caused by T. epicoccoides (G) and T. destructans (H); (I,J) leaf blighted after infection by T. destructans; (K,L) heavy sporulation of T. epicoccoides; (M,N) water-soaked and chlorosis symptoms caused by T. destructans on the adaxial (M) and abaxial (N) leaf surface; and

Figure 1 .
Figure 1.Disease symptoms associated with Teratosphaeria epicoccoides and T. destructans on Eucalyptus plantations in southern China: (A,B) trees in 2-year-old Eucalyptus urophylla × E. grandis plantations associated with T. epicoccoides and T. destructans.Leaves of the whole trees were infected and resulted in intense defoliation in the tree growth season; (C,D) typical leaf spot, vein delimitation, and chlorosis symptoms on leaves of E. urophylla × E. grandis associated with T. epicoccoides.New leaves on the top of shoots emerged after infection (C); (E,F) all leaves of 0.5-year-old E. urophylla × E. grandis trees in one plantation infected by T. destructans; (G,H) typical disease symptoms on E. urophylla × E. grandis leaves caused by T. epicoccoides (G) and T. destructans (H); (I,J) leaf blighted after infection by T. destructans; (K,L) heavy sporulation of T. epicoccoides; (M,N) water-soaked and chlorosis symptoms caused by T. destructans on the adaxial (M) and abaxial (N) leaf surface; and (O-R) Four E. urophylla × E. grandis hybrid genotypes exhibiting chlorosis and leaf spot caused by T. destructans.

Figure 2 .
Figure 2. Map showing the 59 sites in the five provinces in China where the diseased leaf samples were collected, and the Teratosphaeria species identified in each site.

Figure 2 .
Figure 2. Map showing the 59 sites in the five provinces in China where the diseased leaf samples were collected, and the Teratosphaeria species identified in each site.

Figure 4 .
Figure 4. Morphological features of Teratosphaeria destructans: (A-F) curved and occasionally straight conidia with septa from different Eucalyptus trees.(G-K) Conidia with one to three septa: one septum (G); two septa (H,I); and three septa (J,K).

Figure 4 .
Figure 4. Morphological features of Teratosphaeria destructans: (A-F) curved and occasionally straight conidia with septa from different Eucalyptus trees.(G-K) Conidia with one to three septa: one septum (G); two septa (H,I); and three septa (J,K).

Figure 5 .
Figure 5. Phylogenetic tree obtained from the maximum likelihood (ML) analysis of the combined dataset of ITS, tef1, and tub2 in these three gene regions.Bootstrap values ≥ 75% from the ML analysis are indicated at nodes.Isolates reported in this study are highlighted in blue.

Figure 5 .
Figure 5. Phylogenetic tree obtained from the maximum likelihood (ML) analysis of the combined dataset of ITS, tef1, and tub2 in these three gene regions.Bootstrap values ≥ 75% from the ML analysis are indicated at nodes.Isolates reported in this study are highlighted in blue.

Figure 6 .
Figure 6.Phylogenetic tree obtained from the maximum likelihood (ML) analysis of the dataset of the ITS region.Bootstrap values ≥ 75% from the ML analysis are indicated at nodes.Isolates reported in this study are highlighted in blue.

Figure 6 .
Figure 6.Phylogenetic tree obtained from the maximum likelihood (ML) analysis of the dataset of the ITS region.Bootstrap values ≥ 75% from the ML analysis are indicated at nodes.Isolates reported in this study are highlighted in blue.

Figure 7 .
Figure 7. Phylogenetic tree obtained from the maximum likelihood (ML) analysis of the dataset of the tef1 region.Bootstrap values ≥ 75% from the ML analysis are indicated at nodes.Isolates reported in this study are highlighted in blue.

Figure 7 .
Figure 7. Phylogenetic tree obtained from the maximum likelihood (ML) analysis of the dataset of the tef1 region.Bootstrap values ≥ 75% from the ML analysis are indicated at nodes.Isolates reported in this study are highlighted in blue.

Figure 8 .
Figure 8. Phylogenetic tree obtained from the maximum likelihood (ML) analysis of the dataset of the tub2 region.Bootstrap values ≥ 75% from the ML analysis are indicated at nodes.Isolates reported in this study are highlighted in blue.

Figure 8 .
Figure 8. Phylogenetic tree obtained from the maximum likelihood (ML) analysis of the dataset of the tub2 region.Bootstrap values ≥ 75% from the ML analysis are indicated at nodes.Isolates reported in this study are highlighted in blue.

Table 1 .
Location details, collection information, and Eucalyptus tree plantation species of diseased leaf samples collected from 59 sites in five provinces.

Table 2 .
Isolates sequenced and used for phylogenetic analyses in this study.

Table 3 .
Isolates from other studies used in phylogenetic analyses in this study.

Table 4 .
Isolate numbers of each genotype of T. epicoccoides in the Eucalyptus plantations in each of the five provinces.
a The genotype was determined by ITS-tef1-tub2 gene sequences.

Table 5 .
Isolate numbers of each genotype of T. destructans in the Eucalyptus plantations in each of the five provinces.