Morphological characterization and molecular identification of Colletotrichum species associated to sweet persimmon anthracnose..

The highlands of Southern Brazil contribute with 40% of Brazilian persimmon production. Although expanding, persimmon production faces major problems caused by anthracnose disease (black spot), including fruit rot and necrosis of leaves. Several Colletotrichum species (C. horii, C. gloeosporioides, among others) are implicated in persimmon anthracnose around the world. To identify Colletotrichum species associated with persimmon anthracnose in the highlands of Southern Brazil, 34 isolates were analyzed by ITS-rDNA partial region, GAPDH, and TUB2 partial gene sequences, morphological characteristics, and virulence on persimmon fruits and leaves. Data showed a high prevalence of C. horii (85.3%), that associated with its high virulence on fruits and leaves, confirm a considerable degree of host preference. Moreover, other species C. aenigma, C. asianum, C. fructicola, and C. nymphaeae, were identified, but the last three ones exhibited low virulence on fruits and were not able to produce symptoms on leaves. As far as we know this is the first reference on C. asianum in persimmon. The present data may contribute to a better understanding of the etiology of anthracnose in sweet persimmon in Southern Brazil, and it will be useful for epidemiological studies and the development of disease management measures.


INTRODUCTION
Persimmon (Diospyros kaki L.) was introduced in Brazil by Japanese immigrants at the beginning of the 20th century, and its growing area has expanded considerably since the 1920s. Nowadays, Brazil is the fourth world producer of persimmon with a planted area of 8,148 ha, and a production that reaches 157 thousand tons/year (IBGE, 2018). The highlands of Southern Brazil, which account for most of the Brazilian production, are part of a subtropical and temperate macroregion with altitude from 500 to 680 m, a mean annual temperature of 17°C, and a precipitation of 1908 mm/year.
The most prominent persimmon cultivars in Southern Brazil are Fuyu, Giombo, and Kyoto (FIORAVANÇO & PAIVA, 2007). Although, growing at rates that reach 10% per year in Southern Brazil, Andrioli et al. persimmon culture faces a significant problem of increasing anthracnose damage in fruits leaves and branches, caused by Colletotrichum species, which are responsible for important losses in highly infected fields (MAY DE MIO et al., 2015;CARRARO et al., 2019).
Persimmon anthracnose in new twigs appears as small dark elliptic spots that enlarge during time and can coalesce. Leaves infected by Colletotrichum show small circular to elliptic purple to dark brown spots, and fruit anthracnose appears as small sunken and dark spots that progress to larger dark brown lesions (>20 mm) with grey center and orange conidia. Early fruit infections cause premature fruit drop. Moreover, the disease progress during ripening and after harvest, leading to important losses during storage, that can reach 50 to 90% (ZHANG, 2008;DAMM et al., 2010;PALOU et al., 2013).
Aiming to expand the knowledge of anthracnose etiology in southern Brazil, the study characterized Colletotrichum isolates associated with the disease from the region based on the molecular, morphological, and pathogenic analysis and identified the species. Furthermore, the present survey can give subsidies for further epidemiological studies and disease management of this devastating disease.

Samples
Persimmon fruits showing typical symptoms of anthracnose were collected from commercial orchards of the highlands of Southern Brazil (Table 1). Fruit skin tissues of approximately 5 mm in diameter, were collected, surface-sterilized with 1% NaClO for 1 min, washed twice with sterile distilled water, and partially dried with filter paper. The tissues were placed on PDA (potato-dextrose-agar) amended with gentamicin (50 mg/L) and cefotaxime (100 mg/L). The plates were incubated at 27°C for 4 days. Single-spore cultures were maintained in PDA slants and stored at -80°C in cryotubes with 25% glycerol.

DNA extraction and PCR amplifications
DNA was extracted from fungal mycelia using the method proposed by TAPIA-TUSSELL et al. (2006). DNA was quantified by absorbance at 260 nm, and the quality was estimated by 260/280 ratio and agarose gel electrophoresis.

DNA sequencing of the amplicons
Amplification products were enzymatically purified with EXOI/SAP (USB) following manufacture instructions. Purified amplicons (50-60 ng) were sequenced using Big Dye Terminator V.3.1 sequencing kit (Thermo) and analyzed with a 3500 Genetic Analyzer (Thermo). Data were collected by Data Collection software (Thermo).

Molecular classification and phylogenetic analysis
The DNA sequences were compared with those deposited in GenBank using the BLAST similarity test. Alignment of the ITS, GAPDH and, TUB2 sequences of the Colletotrichum isolates, references of Colletotrichum species, and Monilochaetes infuscans (outgroup) retrieved from NCBI database was performed with CLUSTAL X. Concatenated sequences were used for phylogenetic analysis using the maximum parsimony of MEGA 6.0 (TAMURA et al., 2013).
Clade stability was determined by 1,000 bootstrap replicates.

Morphological characteristics of the species
Radial mycelial growth was recorded daily by two perpendicular measures, for 7 days on PDA cultures at 28°C with 16 h photoperiod, initiated from 5 mm diameter mycelium plugs. Colony appearance and conidial production were evaluated on the seventh day. Conidia were recovered after 7 days on PDA plate by scraping with saline, followed by filtration. Conidial shape and size were microscopically evaluated at x 400 magnification using a Nikon E200 microscope coupled with a CCD camera. Conidial appressoria were evaluated as proposed by LIU et al. (2016).

Pathogenicity test
The pathogenicity of representatives of the Colletotrichum species (five species and a total of 11 isolates) isolated from persimmon fruits was evaluated on leaves and fruits of Kyoto cultivar. Intact leaves were inoculated with 0.5 cm plugs of fungal mycelia obtained from 7 days cultures on PDA medium. The necrotic spots diameter was evaluated 7 days after incubation at 28°C on 99% humidity chambers. Persimmon fruits (without lesions) were inoculated with 10 µL conidial suspensions (1 x 10 6 conidia/mL) freshly prepared and incubated on 99% humidity chambers for 7 days. The lesion diameters were determined using a digital calypter. In each assay, a total of eight repetitions (fruit or leaves) were utilized.

Statistical analysis
Results were analyzed statistically by analysis of variance (one-way ANOVA), and Tukey's test. The statistical analyses were performed using IBM-SPSS Statistics -version 22 software, and statistical significance was attributed to values of P ≤ 0.05.

RESULTS AND DISCUSSION
A total of 34 Colletotrichum were isolated from 36 persimmon fruits with symptoms of anthracnose collected on the highlands of Southern Brazil (Table 1). These isolates were all pathogenic to persimmon fruits and 60% of the leaves presented the anthracnose symptoms. Isolates were evaluated by their ITS (514 bp), GAPDH (235 bp), and TUB2 (520 bp) sequences, and classified by comparison with reference sequences deposited on GenBank (Table 2). As can be observed in figure 1, 97% of the isolates were included in the Gloeosporioides clade. Among these, 85.3% (29/34) Table 2 -Identification of Colletotrichum isolates and their GenBank accession code for ITS, GAPDH and TUB2 partial sequences.

Species
Isolate were highly related with Colletotrichum horii reference, 5.9% (2/34) were similar to C. fructicola, one isolate was closely related with C. asianum (2.9%), a sister species of C. fructicola, and one was classified as C. aenigma (2.9%). Just one isolate was classified as C. nymphaeae, a member of the Acutatum clade.
Colletotrichum species associated with anthracnose disease in persimmon varied depending on the classification methods. ZHANG et al. (2008), based on morphological characteristics classified persimmon anthracnose associated fungi as C. gloeosporioides, while WEIR & JOHNSTON (2010) classified New Zealand isolates as a new species C. horii, within the Gloeosporioides clade. The present study, based on phylogenetic characterization, corroborated the high prevalence of C. horii associated with persimmon anthracnose, previously reported in both leaves and fruit diseases of persimmon in New Zealand, China, Korea, Japan, and Brazil (WEIR & JOHNSTON, 2010;YU et al., 2013;KWON et al., 2013;MAY DE MIO et al., 2015;HASSAN et al., 2018;JEON et al., 2017;ASANO & HIRAYAMA, 2019;CARRARO et al., 2019). Colletotrichum horii is highly host-specific (WEIS et al., 2012) which explains its high prevalence even in a region where crops such as apple and grapes, committed by C. fructicola and C. viniferum, are grown side by side with persimmon.
Although in a low-frequency, other species of Colletotrichum were isolated from persimmon fruits with anthracnose symptoms. C. fructicola has been reported as a causal agent of fruit anthracnose in apple (VELHO et al. 2015), grapes (ECHEVERRIGARAY et al., 2020), and other crop fruits in Brazil, and was first reported in persimmon in Parana State (Southern Brazil) by CARRARO et al. (2019), and C. aenigma was previously associated to sweet persimmon anthracnose in Korea (HASSAN et al., 2018), and grape ripe-rot disease in Southern Brazil (ECHEVERRIGARAY et al., 2020). Moreover, C. asianum is associated with anthracnose in coffee (PRIHASTUTI et al., 2009) and mango (SHARMA et al., 2015, VITALE et al., 2020, but this is the first report of this species on persimmon. Just one isolate (2.9%) was classified as C. nymphaeae (Acutatum clade), and references to this species associated with persimmon anthracnose were cited in Korea (HASSAN et al., 2018) andBrazil (CARRARO et al., 2019).
Six representative isolates of C. horii and all the isolates of the other species identified by sequencing were further evaluated for their growing and morphological characteristics. As can be observed in figure 2 and table 3, the Gloeosporioides clade species (C. horii, C. fructicola, C. aenigma, nymphaeae. Colonies on PDA from above (left) and below (middle), and conidia (right). Scale bar: 20 µm. and C. asianum) showed similar growing culture patterns on BDA medium, but the C. nymphaeae isolate exhibited a lower growth rate than the species of Gloeosporioides clade. Although, considered an important character of fungi, the growth rate is not directly related to fungal pathogenicity. As can be observed in figure 2 and table 3, C. horii, and C. asianum showed similar growth color and behavior defined as grey-green, with a reverse light grey with darker grey sectors. Conversely, C. aenigma had white to pale grey colony with concentric pale grey sectors, and C. nymphaeae showed light grey/green colonies with reverse white with concentric grey/ green sectors. The morphological characteristics of the persimmon isolates obtained in this research are similar to those described for these species (WEIS et al., 2012;DAMM et al., 2012).
All isolates produce hyaline conidia with a classical cylindrical shape with broadly rounded ends ( Figure 2 and Table 3). However, as expected, species classified within the Gloeosporioides clade (C. horii, C. fructicola, C. aenigma, and C. asianum) showed longer and wider conidia than those of the C. nymphaeae isolate (Acutatum clade). Moreover, among the Gloeosporioides clade species, the conidia of C. asianum was somehow shorter than the other species of the clade, confirming the data published by WEIR et al. (2012).
Conidial appressoria were produced by all the isolates and species but were particularly scarce in the C. aenigma isolate. All of them exhibited the characteristic light brown to brown color determined by the accumulation of melanin pigments (KUBO & FURUZAWA, 1991). Conidial appressoria showed a similar size (14.1 ± 1.3 x 7.3 ± 1.4 µm) and did not differ significantly within and among species.
Pathogenicity tests on fruits and leaves of persimmon (vr. Kyoto) showed that all the species were able to infect fruits (Figure 3), and C. horii isolates cause larger lesions than the other species, indicating that the aggressiveness to persimmon varied among species, and can be responsible for the high prevalence of C. horii in anthracnose symptomatic fruits. Moreover, the six isolates of C. horii evaluated developed larger necrotic spots on persimmon leaves, reinforcing the high pathogenicity and host-specificity of C. horii on persimmon (XIE et al., 2010;MAY DE MIO et al., 2015). Moreover, just one of the two isolates of C. fructicola, and none of the isolates of C. aenigma, C. asianum, and C. nymphaeae developed necrotic regions on persimmon leaves.    aenigma (n= 1), C. asianum (n=1), and C. nymphaeae (n=1) isolates, all of them with 8 replications, after 7 days of incubation (28°C; 99% humidity). Bars with the same letters were not significantly different by the Tukey test (P ≤ 0.05).
In conclusion, based on morphological data and molecular classification, five species of Colletotrichum were identified as causal agents of anthracnose in persimmon in the Southern Brazil region. These species belong to two different complexes (gloeosporioides and acutatum), with a high prevalence of C. horii. Although, all the species were able to infect and develop typical anthracnose symptoms in fruits of persimmon of Kyoto cultivar, the C. horii isolates were the most virulent, and together with C. fructicola were the only species that infected the persimmon leaves.