Diversity of Colletotrichum Species Causing Apple Bitter Rot and Glomerella Leaf Spot in China

Bitter rot and Glomerella leaf spot (GLS) of apples, caused by Colletotrichum species, are major diseases of apples around the world. A total of 98 isolates were obtained from apple fruits with bitter rot, and 53 isolates were obtained from leaves with leaf spot in the primary apple production regions in China. These isolates were characterized morphologically, and five gene regions (ITS, ACT, GAPDH, CHS-1 and TUB2) were sequenced for each isolate. A phylogenetic analysis, combined with a comparison of the morphological, cultural and pathogenic characters, sorted bitter rot isolates into six species: C. alienum, C. fructicola, C. gloeosporioides sensu stricto, C. nymphaeae, C. siamense and one new species, C. orientalis Dandan Fu & G.Y. Sun. Among these, C. siamense was the predominant pathogen associated with bitter rot. Isolates from leaf spot were identified as two species, C. aenigma and C. fructicola. This is the first report of C. orientalis as an apple bitter rot pathogen worldwide, and the results provide important insights into the diversity of Colletotrichum species in China.

In China, apple bitter rot occurs in almost all producing areas, and the pathogens have been identified as C. gloeosporioides and C. acutatum [33][34][35]. Unfortunately, these species may all represent species complexes. GLS is an emerging disease that was first reported in 2012, and the pathogens have been identified as C. fructicola and C. aenigma [28], yet hidden pathogen diversity may exist due to insufficient investigation. Therefore, the main objective of this study is to investigate the Colletotrichum species diversity associated with ABR and GLS in China; gaining this knowledge will provide clues towards more effective control measures against these devastating diseases.

Isolates
Isolates were collected from diseased apple tissues exhibiting bitter rot and leaf spot symptoms in commercial apple orchards in four provinces, including Liaoning, Shandong, Henan and Shaanxi, of China from 2009 to 2013. Small pieces of symptomatic tissue were cut from lesions, immersed in 70% alcohol for 1 min, rinsed with sterile water and then dried on sterilized filter paper before placement into Petri dishes with Potato Dextrose Agar (PDA, Becton, Dickinson and Company, Franklin Lakes, NJ, USA). Cultures were incubated for 4 days at 25 • C in darkness. A mycelial disc was taken from the actively growing edge of a mono-conidial colony, and then transferred onto new PDA plates. Monosporic isolates were obtained from the new cultures. The surfaces of the PDA plates were scraped with sterile water and collected as conidia suspensions. Monosporic isolates were deposited in the Fungal Laboratory of Northwest A&F University, Yangling, Shaanxi Province, China. After 7 days at 25 • C in darkness, the sizes and shapes of 50 conidia harvested from the cultures were measured and recorded [36]. The colony diameter, color of the conidial masses and zonation of the colony were recorded. Appressoria were induced using a slide culture technique, in which a 1 cm 2 segment of PDA containing the isolate was placed in sterile water in a sterile Petri dish, covered with a sterile coverslip and incubated under high humidity at 25 • C in darkness. After 2 days, the shapes and sizes of 50 appressoria on the coverslip were recorded.

Sequence Alignment and Phylogenetic Analysis
Preliminary alignments of the multi-locus sequences were conducted using Clustal X [44] with a manual adjustment and BioEdit for visual improvement wherever necessary. The concatenation of the five-gene sequences was completed in PhyloSuite [45]. A maximum likelihood (ML) analysis was performed by RAxML version 8 [46] under the GTR model [47], and a non-parametric bootstrap analysis with 1000 repetitions [48] was used to determine the statistical support of the phylogeny. Bayesian inference (BI) phylogeny construction was performed with MrBayes version 3.2.1 [49], with the GTR + G + I nucleotide substitution model. The analysis included two separate runs for 1 × 10 7 generations; each run was sampled every 1000 generations, and the convergence of all the parameters was checked using internal diagnostics. To construct the 50% majority-rule consensus tree, the first 25% generations were discarded as burn-in. The phylogenetic tree ( Figure 1) was visualized using FigTree v 1.4.4. A potential recombination event between C. fioriniae and C. orientalis was detected based on a pairwise homoplasy index (PHI) analysis of the Genealogical Concordance Phylogenetic Species Recognition concept in SplitsTree version 4.11.3 using the multi-locus alignment dataset [50,51].

Sequence Alignment and Phylogenetic Analysis
Preliminary alignments of the multi-locus sequences were conducted using Clustal X [44] with a manual adjustment and BioEdit for visual improvement wherever necessary. The concatenation of the five-gene sequences was completed in PhyloSuite [45]. A maximum likelihood (ML) analysis was performed by RAxML version 8 [46] under the GTR model [47], and a non-parametric bootstrap analysis with 1000 repetitions [48] was used to determine the statistical support of the phylogeny. Bayesian inference (BI) phylogeny construction was performed with MrBayes version 3.2.1 [49], with the GTR + G + I nucleotide substitution model. The analysis included two separate runs for 1 × 10 7 generations; each run was sampled every 1000 generations, and the convergence of all the parameters was checked using internal diagnostics. To construct the 50% majority-rule consensus tree, the first 25% generations were discarded as burn-in. The phylogenetic tree (Figure 1) was visualized using FigTree v 1.4.4. A potential recombination event between C. fioriniae and C. orientalis was detected based on a pairwise homoplasy index (PHI) analysis of the Genealogical Concordance Phylogenetic Species Recognition concept in SplitsTree version 4.11.3 using the multi-locus alignment dataset [50,51]. Bootstrap support values above 60% and Bayesian posterior probability values above 0.9 were given at the nodes. Isolates isolated in this study and type strains are shown in bold. * Ex-holotype, ex-neotype, ex-epitype strains.

Pathogenicity Tests
Twelve representative isolates of Colletotrichum were chosen based on species identity and locations. Healthy apple fruits and leaves were selected, washed with tap water, blown dry in the hood and surface-sterilized with 70% ethanol prior to inoculation. Leaves and fruits were drop-inoculated with the conidia suspension (approximately 10 6 /mL in concentration) or mycelial plugs. The fruits' wounds were made by sterile insect needles with about 10 holes within a circular area of 5 mm in diameter. After inoculation, the fruits were incubated at 25 • C in plastic bags. The disease incidence of each fungal isolate was recorded 3 days after inoculation. For each isolate, at least five fruit/leaf inoculation replicates were performed in each experiment, and the inoculation experiment was repeated two times.  Pueraria lobata Litchi chinensis Japan JX010275 JX009480 JX010020 JX009826 alignment contained a total of 1916 characters, among which 551 were parsimony informative (28.8%). The BI tree, along with both the Bayesian posterior probability values and maximum likelihood bootstrap support values, are shown in Figure 1. The Bayesian tree was identical to the maximum likelihood tree in topology. The phylogram supported eight defined clades, representing C. aenigma, C. alienum, C. fructicola, C. gloeosporioides, C. nymphaeae, C. siamense and a candidate for a new species, respectively. Four isolates clustered with C. hymenocallidis (CBS 125378), and one isolate grouped with C. siamense sensu stricto (CBS 130417 and ICMP 17795), which both belong to C. siamense sensu lato [52]. The clades of C. fructicola (CBS 130416), C. aenigma (ICMP 18608 and, ICMP 18686), C. alienum (ICMP 12071 and ICMP 18621) and C. gloeosporioides (CBS 112999 and CBS 119204) each included nine, three, one and six apple isolates, respectively.
The remaining eight isolates from the diseased apple fruits belonged to the C. acutatum complex. One isolate clustered together with C. nymphaeae (CBS 515.78 and IMI 370491), while the other seven formed a separate clade together with CBS 128555 ( Figure 1). As revealed by the previous multi-locus molecular phylogenetic analysis [12], C. fiorinae contains two well-separated clades; one clade contains CBS 128555, and the other clade contains the type strain CBS 128517. Here, we propose that the CBS 12855 clade should better be defined as an independent taxon unit, which we have named C. orientals. The new species delimitation was also supported by the PHI analysis in which no obvious evidence of recombination was detected between the two clades ( Figure 2).

Phylogenetic Analysis
Based on ITS sequences and cultural characters, 32 representative isolates were chosen for further phylogenic analysis. The five-locus (ITS, ACT, GAPDH, CHS-1 and TUB2) phylogenetic analysis included 51 reference isolates [10,12,27]. Concatenated sequence alignment contained a total of 1916 characters, among which 551 were parsimony informative (28.8%). The BI tree, along with both the Bayesian posterior probability values and maximum likelihood bootstrap support values, are shown in Figure 1. The Bayesian tree was identical to the maximum likelihood tree in topology.
The remaining eight isolates from the diseased apple fruits belonged to the C. acutatum complex. One isolate clustered together with C. nymphaeae (CBS 515.78 and IMI 370491), while the other seven formed a separate clade together with CBS 128555 ( Figure  1). As revealed by the previous multi-locus molecular phylogenetic analysis [12], C. fiorinae contains two well-separated clades; one clade contains CBS 128555, and the other clade contains the type strain CBS 128517. Here, we propose that the CBS 12855 clade should better be defined as an independent taxon unit, which we have named C. orientals. The new species delimitation was also supported by the PHI analysis in which no obvious evidence of recombination was detected between the two clades ( Figure 2).

Taxonomy
Based on the result of multigene phylogeny, the 32 Colletotrichum isolates characterized in this study were grouped into seven species: C. aenigma (three isolates), C. alienum (one isolate), C. fructicola (nine isolates), C. gloeosporioides (six isolates), C. nymphaeae (one isolate), C. siamense (five isolates) and C. orientalis (seven isolates).   Notes: C. aenigma could not be separated from C. alienum by ITS sequence analysis, nor from C. tropicale by ACT. The conidia of the holotype (ICMP 18608) of C. aenigma were (12-)14-15(-16.5) × (5-)6-6.5(-7.5) µm, and the appressoria were subglobose [52], whereas the isolate F12PGXY03 had longer and thinner conidia, and the appressoria were generally oval-shaped and longer than those of ICMP 18608. Additionally, the cultural characters of our isolates were different from those of ICMP 18608. Description: Vegetative hyphae are 1-11 µm diam, hyaline to pale brown, smooth-walled, septate and branched. Conidiophores are hyaline and smooth-walled; a few are septate and branched. Conidiogenous cells are hyaline, smooth, cylindrical and not clearly separated from the hyphae by a septum. Conidia are hyaline, aseptate, straight and cylindrical with both ends rounded or one end slightly acute, (13.1-)14.5-16(-18.5) × (4.5-)5-5.5(-6.2) µm, mean ± SD = 15.38 ± 1.16 × 5.29 ± 0.40 µm (n = 50), L/W ratio = 2.9. Appressoria are single or in loose groups, pale to dark brown, ovoid, cylindrical or fusoid and sometimes slightly irregular, (6-)8.5-11(-13) × (4.4-)5.5-6.5(-8.4) µm, mean ± SD = 9.66 ± 1.74 × 5.94 ± 0.81 µm (n = 50), L/W ratio = 1.6. Colonies on the PDA are 78-80 mm after 7 d. The aerial mycelium is white to pale gray, dense and cottony; in reverse, it is dark gray towards the center and pale gray at the edge. Notes: The ITS sequence analysis did not separate C. fructicola from C. aeschynomenes, and the ACT sequence analysis could not separate it from C. alienum, C. dianesei, C. queenslandicum and C. siamense. Similarly, neither GAPDH nor TUB2 separated this species from C. alienum. The CHS-1 sequence analysis did not separate it from some of the isolates of C. siamense. Nevertheless, these taxa were well-distinguished using multi-gene analysis. Description: Vegetative hyphae are 1-8 µm diam, hyaline to pale brown, smooth-walled, septate and branched. Conidiophores are hyaline to pale brown, smooth-walled and one or two celled; a few are branched. Conidiogenous cells are hyaline, smooth, cylindrical and clearly separated from the hyphae by a septum. Conidia are hyaline, aseptate, straight and cylindrical with both ends rounded, (11.9-)14.5-15.5(-18.9) × (4-)4.5-5(-5.5) µm, mean ± SD = 15.08 ± 1.14 × 4.87± 0.31 µm (n = 50), L/W ratio = 3.1. Appressoria are single or in loose groups, pale to dark brown, ovoid and subglobose or short, mean ± SD = 9.79 ± 1.60 × 5.94 ± 0.77 µm (n = 50), L/W ratio = 1.6. Colonies on the PDA have an entire margin. The aerial mycelium is white, and the colonies are dense, cottony, white to pale gray or dark gray. Occasionally, orange conidial ooze is visible in the center; in reverse, it is buff with sporadic dark gray spots or grayish dark towards the center and pale gray at the edge. Colonies on the PDA are 68-75 mm after 7 d. Notes: After Prihastuti et al. separated Colletotrichum siamense from the C. gloeosporioides complex, more isolates from multiple hosts were identified as this species [54]. ITS sequences separated C. siamense well from other taxa, but the ACT sequence did not separate it from C. alienum, C. hymenocallidis, C. queenslandicum or C. fructicola. Similarly, GAPDH and TUB2 do not separate this species from C. hymenocallidis. C. hymenocallidis was first introduced by Yang et al. from Hymenocallis americana [57] but was recently synonymized with C. siamense [52]. However, Sharma et al. considered C. siamense to be a species complex based on an ApMat sequence analysis because C. siamense showed high sequence variability [58]. Moreover, Liu et al. indicated that more isolates need to be included to support further splitting of C. siamense, which possibly resurrects C. hymenocallidis [59].   Notes: Freeman and Shabi studied isolates from fruit rot of apples and peaches (based on the ITS sequence, probably identifiable as C. fioriniae), which produced lesions Mycobank: MB 808171. Etymology: Referring to the isolates collected from the eastern region of China. Description: Vegetative hyphae are 1-6 µm, hyaline, smooth-walled, septate and branched. Conidiophores are formed directly on the hyphae. Conidiophores are hyaline, smooth-walled, simple or septate and branched. Conidia are hyaline, smoothwalled, aseptate, straight and fusiform or cylindrical with both ends acute, (12.8-)14-16(-18.5) ×(3.9-)4-5(-5.5) µm, mean ± SD = 15.07 ± 1.23 × 4.51 ± 0.38 µm (n = 50), L/W ratio = 3.3 µm. Appressoria are single or in loose groups, pale-to-medium brown, smooth, oval-shaped and ellipsoidal or irregularly outlined, (7-)8-9.5(-11.5) × (4.4-)5.5-6(-7.2) µm, mean ± SD = 8.74 ± 0.99 × 5.84 ± 0.53 µm (n = 50), L/W ratio = 1.5. Colonies on the PDA have an entire margin and are compacted cottony to felty. They are orangish red towards the center and pale gray towards the edge. The aerial mycelium is white to pale gray, and the conidiomata are sparse with masses of orange conidia. In reverse pale brownish pink. Colonies on the OA have an entire margin. The aerial mycelium is sparse, white to pale gray, and on the surface with visible masses of orange conidia scattered in circles; in reverse, it is pale buff. Colonies on the PDA are 45-51 mm after 7 d (67-75 mm in 10 d).
Holotype Notes: Freeman and Shabi studied isolates from fruit rot of apples and peaches (based on the ITS sequence, probably identifiable as C. fioriniae), which produced lesions on many different fruits, indicating that isolates of this group have the ability to cross-infect fruit from multiple hosts [60]. In this paper, we isolated seven isolates from apple bitter rot in Liaoning Province. A phylogenetic analysis (Figure 1) showed that they constituted a monophyletic clade together with the six C. fioriniae isolates (CBS 129938, CBS200.35, ATCC 28992, CBS 119293, CBS 128555 and CBS 490.92). In Damm et al. [12], the clade was well-separated from the clade containing the C. fioriniae holotype CBS 128517. Separation between the two clades was also evident in Damm et al. [12], which was treated as intraspecific heterogeneity. In this study, the PHI analysis detected no significant evidence of recombination between the two clades ( Figure 2). Therefore, we denominate the clade containing the ABR isolates a new species.

Pathogenicity Tests
In the fruit infection assays, the isolates isolated from apples with bitter rot symptoms were pathogenic to the apple fruits in both the unwounded and wounded inoculations ( Table 2). Dark brown rot lesions, similar in appearance, were produced in all cases ( Figure 5). Of the non-wounded inoculations, Colletotrichum alienum (F11PGZH02) had the highest infection incidence (100%), whereas the isolates of C. gloeosporioides (F11PGQX17) and C. orientalis (F10PGBYS08) had the lowest incidences (33%); the others were in the middle. Of the wounded inoculations, all isolates had a 100% infection incidence. Lesions incurred by different isolates were similar in size, except that the lesions incurred by C. nymphaeae (F10PGBYS12, belonging to the C. acutatum complex) were apparently smaller ( Figure 5).    Isolates isolated from the GLS lesions caused GLS lesions on both the apple fruits and leaves ( Table 3). The isolates of C. aenigma (F12PGXY03, W12PGYXY15) and C. fructicola (F12PGSQ0503, W12PGYSQ06) were pathogenic on the leaves and fruits of Gala apples, but non-pathogenic on Fuji apple leaves or fruits in the non-wounded inoculation ( Figure 5), which is in accordance with the observation that Fuji apples are resistant to GLS disease [61]. Table 3. Pathogenicity test of selected isolates on apple leaves.

Species
Isolate Origin Inoculation Cultivar

Discussion
China is the largest apple-producing country in the world. Bitter rot has been a common disease in almost all apple production areas and can cause large economic losses under disease-favorable temperature and humidity conditions. Glomerella leaf spot (GLS) has been a severe foliar disease on cvs. Gala, Jonagold and Golden Delicious in the USA and Brazil. It was found first in Henan, China, in 2010 [28]. Now, it has become prevalent in all major apple-producing areas in China. Thus far, however, the species diversity of apple Colletotrichum pathogens in China is largely unclear. In this study, we collected and characterized 151 isolates from four apple-producing provinces and identified six known species, as well as one new species, demonstrating that diverse Colletotrichum species can infect apples. Moreover, C. orientalis was shown for the first time to be an apple Colletotrichum pathogen.
Among the identified species, C. siamense, C. fructicola, C. aenigma, C. alienum and C. gloeosporioides belong to the CGSC, while C. orientalis and C. nymphaeae belong to the CASC. Overall, the CGSC species appear to be more prevalent compared with the CASC species. Moreover, fruit isolates and leaf isolates differ significantly in their genetic makeups. Seven species were recognized as isolates from apple fruits, whereas only two (C. fructicola and C. aenigma) were recognized as leaf spot isolates. In a pathogenicity test, ABR isolates fail to incur GLS symptoms, and the GLS isolates fail to incur ABR symptoms, indicating a pathogenic differentiation among the two groups of pathogens. Such results are in accordance with a previous study that demonstrated the intraspecific differentiation in the pathogenicity of GLS and ABR for C. fructicola [62].
C. siamense is a species that includes members from diverse hosts and that has a worldwide distribution. The diversity is so high that there has been controversy regarding whether it should be treated as a single species or a species complex. In a recent study carried out by Liu and others [63], six independent species very close to C. siamense s. str. (C. communis, C. hymenocallidis, C. dianesei, C. endomangiferae, C. jasmini-sambac and C. murrayae) were renamed as C. siamense. In this study, the four characterized isolates clustered together with C. hymenocallidis, and one isolate clustered with C. siamense s. str. Based on broad species criteria, these isolates should all be regarded as C. siamense sensus lato. C. fructicola represents another important pathogen species identified in this study. C. fructicola has a very broad host range, having been isolated from over eight plant families as endophytes and as plant pathogens. In this study, C. fructicola was isolated from both bitter rot and Glomerella leaf spot lesions. C. fructicola causes Glomerella leaf spot in Brazil but has been more commonly identified as a bitter rot pathogen in central USA, Brazil and Uruguay. In Uruguay in particular, most isolates from apple bitter rot were identified as C. fructicola. Interestingly, despite the prevalence of C. fructicola in Uruguay, Glomerella leaf spot disease does not occur in the field [62]. In the future, it would be interesting to determine whether there are distinctive C. fructicola populations for isolates from leaf lesions and fruit lesions in China.
In a previous study [12], C. fiorinae has been defined as a species with two wellseparated clades. We propose here that the two clades should be regarded as different species due to the fact that the pairwise homoplasy index (PHI) analysis in SplitsTree did not detect evidence of recombination between them. Therefore, we have named the new lineage C. orientalis. C. alienum and C. gloeosporioides are two other species common in fruits. Interestingly, C. alienum has only been reported in New Zealand and Australia, and C. gloeosporioides has never been reported on apples in China. The identification of these species on apples in China highlights the importance of a diversity survey.
Compared with apple bitter rot, GLS is a relatively new disease. Velho and others have suggested that GLS pathogens originate from apple bitter rot pathogens [62]. In this study, all isolates caused fruit rot upon wound inoculation, whereas only the isolates from the leaf spot lesions incurred leaf spot symptoms. Importantly, such pathogenic differentiation could occur in the same species (e.g., F12PGSQ01 and W12PGYSQ06). Such a pathogenicity differentiation pattern is in line with the report by Velho and others [62].
Isolates showing an intraspecific pathogenic variation would be valuable resources for comparative studies aiming to dissect the mechanisms that underlie the adaptive evolution of apple Colletotrichum pathogens.
In summary, based on a systemic survey of Colletotrichum isolates, in this study, we identified seven species associated with the GLS and ABR diseases in China, highlighting the rich species diversity of Colletotrichum spp. on apples. There are tens of apple-producing provinces in China that are variable in terms of climate and soil conditions; further survey efforts into the hidden species diversity and the structural variations among the different regions is critical for effective control of these two important diseases.