Colletotrichum species associated with loquat anthracnose in Kagawa and Tokushima prefectures, Japan

doi:10.21203/rs.3.rs-3551407/v1

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Colletotrichum species associated with loquat anthracnose in Kagawa and Tokushima prefectures, Japan

https://doi.org/10.21203/rs.3.rs-3551407/v1

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Anthracnose caused by Colletotrichum species is one of the serious diseases of loquat, but diversification of the species of Colletotrichum associated with loquat in Japan has not been adequately surveyed. In this study, 46 isolates were isolated from diseased leaves and fruits of loquat in Kagawa and Tokushima prefectures in 2017 and 2018. Using a combination of morphological features and molecular approaches, seven Colletotrichum species were identified: C. fioriniae, C. nymphaeae, C. karsti, C. aenigma, C. fructicola, C. gloeosporioides s. s., and C. siamense. The most prevalent species was C. karsti followed by C. fioriniae and C. gloeosporioides s. s., accounting for 43.5, 21.7, and 13.0%, respectively. All identified species were confirmed to be the causal agents of loquat anthracnose by applying Koch’s postulates. Moreover, potential cross-infection from loquat to kiwifruit, satsuma mandarin, persimmon, and peach was found, in which loquat isolates of C. fioriniae, C. fructicola, and C. siamense caused symptoms on all the tested plants. This report is the first on C. karsti and C. aenigma associated with loquat anthracnose and the first record of C. fructicola and C. gloeosporioides s. s. in Japan, providing crucial information for epidemiology.

Colletotrichum spp.

Eriobotrya japonica

multilocus phylogeny

cross-infection

Loquat (Eriobotrya japonica (Thunb.) Lindl) is an ancient subtropical evergreen fruit tree originating in China (Tian et al. 2011). Loquat fruit is rich in essential dietary nutrients including sugars, protein, carotenoids, fiber, vitamins A and C, and minerals (Tian et al. 2011; Li et al. 2016). Loquat leaves are rich in fiber, minerals, and vitamins B2, B6, and B12, and have several medicinal properties such as antioxidant, antidiabetic, antitumoral, anti-inflammatory, antinociceptive, and antimicrobial (Cha et al. 2011; Khouya et al. 2022; Kuraoka-Oliveira et al. 2020; Shen et al. 2021). These properties lead loquat to be commercially cultivated in more than 20 countries including China, India, Australia, Brazil, Italy, Spain, Turkey, and Japan (Tian et al. 2007, 2011). In Japan, loquat is a minor crop with about 2,500 tons harvested in 2022 from 905 ha (Ministry of Agriculture, Forestry and Fisheries; https://www.maff.go.jp/index.html). Nevertheless, the appearance of foliar and fruit diseases has been found annually in loquat cultivation, with little attention being paid to disease management.

More than 200 fungal species have been associated with loquat worldwide (Farr and Rossman 2023). Among them, anthracnose caused by Colletotrichum species is one of the serious diseases that damages fruit quality and is considered the major postharvest disease of loquat fruit (Cao et al. 2008; Palou et al. 2016). Infected fruits show brown sunken lesions, which later turn into black, hard, and shriveled mummies (Naz et al. 2017). The fungal pathogen can infect leaves, which causes small brown to reddish-brown necrotic spots or sunken lesions (Naz et al. 2017; Kuang et al. 2021). Under high humidity, the whole fruit may rot, and fungal fruiting bodies with pale orange to orange spore masses form on the rotten surface (Cao et al. 2008; Cao and Zheng 2010; Liu et al. 2007).

The genus Colletotrichum was first introduced by Corda (1831) and is the sole member of the family Glomerellaceae (Glomerellales, Sordariomycetes) (Maharachchikumbura et al. 2016; Hyde et al. 2016, 2020). This genus comprises more than 248 accepted species belonging to 14 species complexes and 13 singletons (Jayawardena et al. 2021). Many are important phytopathogens, and some are endophytes and saprobes (Cannon et al. 2012; Hyde et al. 2014, 2020; Jayawardena et al. 2021). In loquat, seven species of Colletotrichum have been recorded including C. fioriniae, C. nymphaeae, C. godetia, and C. eriobotryae from the C. acutatum species complex (CASC) and C. fructicola, C. gloeosporioides s. s., and C. siamense from the C. gloeosporioides species complex (CGSC) (Damm et al. 2020; Farr and Rossman 2023; James et al. 2014; Juárez‑Vázquez et al. 2019; Kuang et al. 2021; Naz et al. 2017; Sato et al. 2013). However, only C. fioriniae, C. nymphaeae, and Colletotrichum sp. have been recorded in Japan, and these species were last isolated more than a decade ago (NARO Genebank, https://www.gene.affrc.go.jp/databases-micro_search_en.php?pldis=5601). Therefore, the species diversification of Colletotrichum in Japan may have changed.

In Kagawa Prefecture, Japan, loquat is cultivated in orchards, in-house, by the roadside, and near other important economic fruit crops such as kiwifruit, satsuma mandarin, persimmon, and peach. According to several reports, some Colletotrichum species have a wide host range, and the phenomenon of cross-infection from the original host to other hosts has been reported (de Aguiar Carraro et al. 2022; Eaton et al. 2021; Jayawardena et al. 2016; Moral et al. 2021; Talhinhas and Baroncelli 2021). This finding leads to a concern that cross-infection by Colletotrichum species might occur between loquat and other proximately cultivated fruit crops. If cross-infection does occur, loquat might be an inoculum source and an alternate host for the overwintering of pathogens, indicating the necessity to pay attention to disease management.

Therefore, this study aimed to identify the Colletotrichum species associated with loquat in Kagawa and Tokushima prefectures based on morphological characteristics and phylogenetic analysis and evaluate the potential for cross-infection by isolates from loquat to kiwifruit, satsuma mandarin, persimmon, and peach.

Sample collection, fungal isolation, and morphological examination

Diseased fruits and leaves of loquat were collected from orchards in Kagawa and Tokushima prefectures, Japan, in 2017 and 2018. A segment (5 × 5 mm) was cut from the boundary of diseased and healthy tissue using a sterilized scalpel. The tissue was surface sterilized by immersion in 1% sodium hypochlorite for 1 min followed by rinsing three times in sterilized distilled water to remove the sterilizing reagent. After drying by blotting with a sterilized paper towel, the tissue was placed in the center of a Petri dish containing potato dextrose agar (PDA; Nissui Pharmaceutical, Tokyo, Japan), which was then incubated at 25°C for 3–5 days in the dark. The hyphal tips cut from mycelia growing from the tissue were transferred to a new Petri dish containing PDA and incubated at 25°C for 7–14 days in the dark. Conidia were harvested to prepare a conidial suspension for purifying the isolate by single spore isolation. Cultural and morphological characterization was examined following the method described previously (Cai et al. 2009; Yu et al. 2022). For long-term storage, a conidia suspension in 25% glycerol of individual pure culture was maintained at − 80°C.

DNA extraction, PCR amplification, and sequencing

The genomic DNA of the isolate was extracted using the CTAB extraction method with slight modifications (Lee et al. 1988; Zhang et al. 2010) and finally eluted with nucleic acid-free water. The DNA was quantified using a NanoDrop1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), and the concentration adjusted to 50 ng/µl to use as the template for PCR reactions. The sequence of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene of all the isolates was amplified and sequenced using primers GDF1 and GDR1 (Templeton et al. 1992). Furthermore, representative isolates were selected to amplify the sequences of the internal transcribed spacer (ITS) region of rDNA, ß-tubulin (TUB2), actin (ACT), chitin synthase 1 (CHS-1), and calmodulin (CAL, for the species belonging to CGSC and CBSC) genes using primer pairs ITS1/ITS4 (White et al. 1990), T1/Bt2b (O’Donnell and Cigelnik 1997; Glass and Donaldson 1995), ACT512F/ACT783R, CHS-79F/CHS-345R (Carbone and Kohn 1999), and CL1C/CL2C (Weir et al. 2012), respectively. PCR was performed under the conditions described by Poti et al. (2023). The PCR products were purified using the PCR clean-up Gel extraction kit (Macherey-Nagel GmbH & Co. KG, Düren, Germany) and sequenced in both forward and reverse directions by Fasmac Co. Ltd. (Kanagawa, Japan). The consensus sequences generated in this study were deposited in GenBank (Table 1).

Table 1

GenBank accession numbers of the *Colletotrichum* isolates generated in this study
Species	Isolate	Host	GenBank accession number^a
Species	Isolate	Host	GAPDH	ITS	TUB2	ACT	CHS-1	CAL
Colletotrichum fioriniae	KMI-2-2-Ca1	Eriobotrya japonica cv. Mogi	OQ440351	OQ430706	OQ440361	OQ440324	OQ440341	-
	KMI-2-2-Ca2	E. japonica cv. Mogi	OQ440352	OQ430707	OQ440362	OQ440325	OQ440342	-
C. nymphaeae	TSK-1-Cg2	E. japonica cv. Nagasakiwase	OQ440358	OQ430713	OQ440368	OQ440331	OQ440348	-
C. karsti	YMZ-2-1-Cg1	E. japonica	OQ440349	OQ430704	OQ440359	OQ440322	OQ440339	OQ440332
	NB-2-2-Cg1	E. japonica	OQ440350	OQ430705	OQ440360	OQ440323	OQ440340	OQ440333
	TI-1-1-2-Cg2	E. japonica cv. Mogi	OQ440356	OQ430711	OQ440366	OQ440329	OQ440346	OQ440337
C. aenigma	STO-2-2-2-Cg2	E. japonica cv. Mogi	OQ440357	OQ430712	OQ440367	OQ440330	OQ440347	OQ440338
C. fructicola	ZYH-3-1-2-Ca1	E. japonica	OQ440354	OQ430709	OQ440364	OQ440327	OQ440344	OQ440335
C. gloeosporioides s. s.	ZYH-1-3-2-Cg1	E. japonica cv. Mogi	OQ440353	OQ430708	OQ440363	OQ440326	OQ440343	OQ440334
C. siamense	FuchuS-3-Cg2	E. japonica cv. Mogi	OQ440355	OQ430710	OQ440365	OQ440328	OQ440345	OQ440336
^aGAPDH = glyceraldehyde-3-phosphate dehydrogenase, ITS = internal transcribed spacer region of ribosomal DNA, TUB2 = β-tubulin, ACT = actin, CHS-1 = chitin synthase 1, and CAL = calmodulin

Phylogenetic analysis

Multilocus phylogenetic analysis based on Bayesian inference (BI) was separately conducted for the three species complexes. Five loci (ITS, GAPDH, CHS-1, ACT, and TUB2) were used to generate a concatenated dataset for the C. acutatum species complex (Damm et al. 2012a); six loci (ITS, GAPDH, CHS-1, ACT, TUB2, and CAL) were used for the C. boninense species complex (Damm et al. 2012b); and six loci (ACT, TUB2, CAL, CHS-1, GAPDH, and ITS) were used for the C. gloeosporioides species complex (Jayawardena et al. 2016). The sequences of type isolates of each species used for the analyses were gained from GenBank and are listed in Supplementary Tables S1 to S3. Outgroups for the C. acutatum, C. boninense, and C. gloeosporioides species complex analyses were Colletotrichum orchidophilum Allesch. (CBS 632.80), C. gloeosporioides s. s. (CBS 112999), and C. boninense Moriwaki, Toy. Sato & Tsukib. (CBS 123755), respectively. Multiple sequences were aligned using MAFFT v.7 (Katoh et al. 2019), combined, and manually improved where necessary using BioEdit v.7 (Informer Technologies, Inc., California, USA). BI analysis was carried out through the CIPRES Science Gateway v.3.3 (Miller et al. 2010; https://www.phylo.org) with the parameters described by Tan et al. (2022). The phylogenetic trees were illustrated using FigTree v.1.4.3 (http://tree.bio.ed.ac.uk/software/figtree/) and edited in Adobe Illustrator 2022 (Adobe Inc., San Jose, California, USA).

Pathogenicity test

The representative isolate of each Colletotrichum sp. was used in pathogenicity tests on detached fruits of the loquat cultivar ‘Mogi’. Moreover, detached fruits of persimmon (Diospyros kaki cultivar ‘Fuyu’), kiwifruit (Actinidia chinensis cultivar ‘Sanuki Angle Sweet’), peach (Prunus persica cultivar ‘Hashiba-hakuho’), and satsuma mandarin (Citrus unshiu cultivar ‘Aoshima Unshu’) were used to assess the potential for cross-infection. The healthy fruits of these plants were harvested from experimental fields of the Fuchu Fruit Research Institute, Kagawa, Japan. The fruits were rinsed with sterilized distilled water several times and air-dried. Two inoculation sites (intact and wounded) were set for each fruit, and three fruits of each plant were used per isolate. To create a wound, a sterilized needle was inserted into the fruits to a depth of 3 mm. A mycelial plug cut from the margin of a 7-day-old colony was placed upside down on the inoculation site, while control fruits were inoculated with a PDA plug. The inoculated fruits were placed in a moist plastic box and incubated at 25°C for 7 days. Symptoms were recorded every day and photographed at 7 days of inoculation. The experiment was conducted twice.

Fungal isolation and morphological characterization

A total of 46 isolates were isolated from the symptomatic leaves and fruits of loquat in Kagawa and Tokushima prefectures, Japan, of which 40 were from the leaves and 6 were from the fruits. Based on culture and conidial characteristics, the isolates were classified into 8 morphotypes (Fig. 1, Table 2). The highest number of isolates found were morphotype III with 11 isolates followed by morphotypes I, IV, VII, and VIII with 10, 9, 6, and 4 isolates, respectively. The least common were morphotypes II, V, and VI with only 2 isolates each. The isolates with morphotypes I and II producing fusiform conidia with both ends acute were assigned to the Colletotrichum acutatum species complex (CASC), while isolates with morphotypes III and IV producing cylindrical conidia with a prominent hilum were assigned to the C. boninense species complex. The isolates with morphotypes V to VIII producing cylindrical conidia were assigned to the C. gloeosporioides species complex.

Table 2

Morphological characteristics of the *Colletotrichum* species causing kiwifruit anthracnose in Japan
Morphotype	Isolate name^a	Colony character on PDA	Conidia	Appressoria
Type I (C. fioriniae)	KMI-2-2-Ca1 KMI-2-2-Ca2 FuchuB-1-1-Ca1 FuchuB-1-1-Ca2 Yuta1-Ca1 Yuta1-Ca2 Yuta4-Ca1 Yuta4-Ca2 Yuta7-1-Ca1 Yama4-3-Ca1	Cottony, reddish colony with white to olivaceous gray aerial mycelia dense at the center. Reverse: reddish. Orange conidial masses were observed to be randomly distributed but dense at the center. Growth rate = 8.8 and 8.7 mm/day	Hyaline, smooth-walled, aseptate, straight, ellipsoid to fusiform, both ends acute with size of 13.0 ± 0.9 × 4.5 ± 0.3 µm (L/W ratio = 2.9) or 12.9 ± 1.0 × 4.6 ± 0.4 µm (L/W ratio = 2.8)	Sepia to medium brown, smooth-walled, globose to ovate, 16.6 ± 2.5 × 12.1 ± 1.5 µm (L/W ratio = 0.7) or 9.0 ± 1.2 × 6.8 ± 0.9 µm (L/W ratio = 0.8)
Type II (C. nymphaeae)	TSK-1-Cg1 TSK-1-Cg2	White to light pale gray aerial mycelia dense at the center. Reverse: pale orange to olivaceous gray. Orange conidial masses were observed at the center of the colony. Growth rate = 9.2 mm/day	Hyaline, smooth-walled, aseptate, straight, ellipsoid to fusiform, both ends sharp with size of 14.8 ± 1.8 × 4.7 ± 0.8 µm (L/W ratio = 3.1)	Medium brown, smooth-walled, globose to ovate, 10.9 ± 2.3 × 7.3 ± 1.1 µm (L/W ratio = 0.7)
Type III (C. karsti)	YMZ-2-1-Cg1 NB-2-2-Cg1 ZYH-1-1-2-Cg1 ZYH-1-1-2-Cg2 ZYH-2-3-Cg2 ZYH-3-1-1-Cg1 ZYH-3-1-1-Cg2 ZYH-3-2-2-Cg1 ZYH-3-2-2-Cg2 Yuta7-1-Cg1 Yuta7-1-Cg2	White aerial mycelium. Reverse: pale yellow. Growth rate = 9.0 and 9.7 mm/day	Hyaline, smooth-walled, aseptate, straight, cylindrical with rounded ends and a hilum-like base, 14.3 ± 0.9 × 5.9 ± 0.4 µm (L/W ratio = 2.4) or 13.5 ± 1.1 × 6.2 ± 0.7 µm (L/W ratio = 2.2)	Sepia to medium brown, smooth-walled, sometimes lobed, globose to ovate, 8.8 ± 1.3 × 6.5 ± 0.9 µm (L/W ratio = 0.7) or 8.8 ± 1.3 × 6.2 ± 0.9 µm (L/W ratio = 0.7)
Type IV (C. karsti)	YMZ-2-1-Cg3 NB-2-2-Cg2 ZYH-2-3-Cg1 TI-1-1-1-Cg1 TI-1-1-1-Cg2 TI-1-1-2-Cg1 TI-1-1-2-Cg2 Tob-2-2-1-Cg1 Tob-2-2-1-Cg2	Thin white aerial mycelium with randomly distributed orange spore masses. Reverse: light yellow with orange spots of conidial masses. Growth rate = 10.2 mm/day	Hyaline, smooth-walled, aseptate, straight, cylindrical, rounded ends with a hilum-like base, 14.9 ± 0.9 × 6.7 ± 0.7 µm (L/W ratio = 2.2)	Sepia to medium brown, smooth-walled, sometimes lobed, globose to ovate, 8.1 ± 0.9 × 6.4 ± 0.6 µm (L/W ratio = 0.8)
Type V (C. aenigma)	STO-2-2-2-Cg1 STO-2-2-2-Cg2	Olivaceous gray colony with flat white to pale gray aerial mycelia. Reverse: Olivaceous gray to brown. Growth rate = 12.2 mm/day (calculated at 5 d)	Hyaline, smooth-walled, aseptate, straight or slightly distorted, cylindrical with one slightly acute or broadly rounded ends, 14.2 ± 1.5 × 4.1 ± 0.5 µm (L/W ratio = 3.5)	Sepia to medium brown, irregular, lobed, 9.5 ± 1.4 × 7.1 ± 0.7 µm (L/W ratio = 0.8)
Type VI (C. fructicola)	ZYH-3-1-2-Ca1 ZYH-3-1-2-Ca2	Olivaceous gray colony with pale gray aerial mycelia. Reverse: olivaceous gray to dark gray, dark at the center of colony. Growth rate = 12.2 mm/day (calculated at 5 d)	Hyaline, smooth-walled, aseptate, cylindrical with rounded ends, 13.0 ± 1.0 × 4.3 ± 0.5 µm (L/W ratio = 3.0)	Sepia to medium brown, smooth-walled, ovate, 9.4 ± 1.6 × 7.4 ± 1.2 µm (L/W ratio = 0.8)
Type VII (C. gloeosporioides s. s.)	ZYH-1-3-2-Cg1 ZYH-1-3-2-Cg2 ZYH-3-3-2-1-Ca1 ZYH-3-3-2-1-Ca2 Yuta3-1-Cg1 Yuta3-1-Cg2	Cottony, white to pale gray aerial mycelia. Reverse: olivaceous gray, dark at the center. Growth rate = 10.2 mm/day	Hyaline, smooth-walled, aseptate, straight cylindrical with rounded ends, 14.4 ± 1.0 × 4.6 ± 0.6 µm (L/W ratio = 3.2)	Sepia to medium brown, smooth-walled, ovate to clavate, 8.6 ± 1.4 × 6.2 ± 0.7 µm (L/W ratio = 0.7)
Type VIII (C. siamense)	FuchuS-3-Cg1 FuchuS-3-Cg2 TI-1-2-Ca1 TI-1-2-Ca2	Cottony, white aerial mycelia and white on the reverse side. Growth rate = 11.6 mm/day	Hyaline, smooth-walled, aseptate, cylindrical with rounded ends, 13.4 ± 0.8 × 4.9 ± 0.5 µm (L/W ratio = 2.7)	Sepia to medium brown, smooth-walled, ovate, 9.1 ± 1.4 × 6.8 ± 0.8 µm (L/W ratio = 0.8)
^a Isolate name in bold was selected as representative isolate for describing morphological characteristics and for phylogenetic analyses.

Phylogenetic analysis

Preliminary analysis of the GAPDH sequences of all the isolates using a BLAST search in the NCBI nucleotide database (https://www.ncbi.nlm.nih.gov) showed that the isolates with morphotypes I to VIII were similar to Colletotrichum fioriniae R.G. Shivas & Y.P. Tan, C. nymphaeae (Pass.) Aa, C. karsti You L. Yang, Zuo Y. Liu, K.D. Hyde & L. Cai, C. karsti, C. aenigma B.S. Weir & P.R. Johnst., C. fructicola Prihast., L. Cai & K.D. Hyde, C. gloeosporioides (Penz.) Penz. & Sacc. sensu stricto, and C. siamense Prihast., L. Cai & K.D. Hyde, respectively. To confirm the results, multiple loci phylogenetic analyses of the representative isolates from each morphotype were conducted. The phylogram of the CASC shows isolates KMI-2-2-Ca1 and KMI-2-2-Ca2 (morphotype I) are clustered with C. fioriniae (CBS 127601 and CBS 119186), and isolate TSK-1-Cg1 (morphotype II) is clustered with C. nymphaeae (CBS 125996, CBS 231.49, and CBS 515.78), with Bayesian posterior probabilities of 1 for all (Fig. 2a). The CBSC phylogram shows isolates YMZ-2-1-Cg1, NB-2-2-Cg1 (morphotype III), and TI-1-1-2-Cg2 (morphotype IV) are clustered with C. karsti (Bayesian posterior probabilities = 1) (Fig. 2b). The CGSC phylogram shows isolates STO-2-2-2-Cg2 (morphotype V), ZYH-3-1-2-Ca1 (morphotype VI), ZYH-1-3-2-Cg1 (morphotype VII), and FuchuS-3-Cg2 (morphotype VIII) are clustered with C. aenigma, C. fructicola, C. gloeosporioides, and C. siamense, respectively, with Bayesian posterior probabilities of 1 for all (Fig. 2c). These results confirmed that seven species of Colletotrichum were isolated from loquat, of which C. karsti was the most prevalent in the population (20 isolates).

Pathogenicity on loquat fruit and cross-infection on the other fruits

A pathogenicity test on the loquat fruits showed that all the isolates caused anthracnose symptoms at the wounded site. Only C. nymphaeae (TSK-1-Cg2), C. fructicola (ZYH-1-3-2-Ca1), and C. siamense (FuchuS-3-Cg2) produced symptoms at both the wounded and intact sites; however, the lesions on the wounded sites were larger than on the intact sites (Fig. 3, Table 3). The symptoms were brown sunken necrotic lesions, and the surrounding tissue was abnormally soft. Symptoms caused by the different isolates were indistinguishable, except the fruit inoculated with C. fructicola had dark, visible fruiting bodies. No symptoms appeared on the control fruits. Moreover, the fungus could be successfully re-isolated from the inoculated fruits and had morphological features similar to the original isolate. These results satisfied Koch’s postulates and verified that all seven species of Colletotrichum identified in this study are the causal agent of loquat anthracnose.

Table 3

Pathogenicity test of the representative isolates of *Colletotrichum* on loquat, kiwifruit, satsuma mandarin, persimmon, and peach fruits.
Species	Isolate	Loquat^a		Kiwifruit		satsuma mandarin		Persimmon		Peach
Species	Isolate	W^b	I	W	I	W	I	W	I	W	I
C. fioriniae	KMI-2-2-Ca2	++	–	++	–	+	–	+	+	+	+
C. nymphaeae	TSK-1-Cg2	++	+	+	–	+	–	+	–	–	–
C. karsti	YMZ-2-1-Cg1	++	–	+	–	–	–	+	–	–	–
C. aenigma	STO-2-2-2-Cg2	++	–	+	–	–	–	+	–	–	–
C. fructicola	ZYH-3-1-2-Ca1	++	+	++	–	+	–	+	+	++	+
C. gloeosporioides s. s.	ZYH-1-3-2-Cg1	++	–	+	–	–	–	+	–	–	–
C. siamense	FuchuS-3-Cg2	++	+	++	–	+	–	+	–	++	++
^a – indicates no symptom developed, + indicates a small lesion, ++ indicates a lesion larger than the inoculation site. ^b W and I are wounded and intact sites, respectively

Inoculation tests on the fruits of kiwifruit, satsuma mandarin, persimmon, and peach were conducted. The results showed that C. fioriniae (YMZ-2-1-Cg1) caused the symptoms on all the tested plants, and severe symptoms were observed on kiwifruit at the wounded sites (Fig. 4, Table 3). Colletotrichum fructicola (ZYH-1-3-2-Ca1) and C. siamense (FuchuS-3-Cg2) also produced symptoms on all the tested plants, but severe symptoms were observed on kiwifruit and peach at the wounded sites. Colletotrichum karsti (YMZ-2-1-Cg1), C. aenigma (STO-2-2-2-Cg2), and C. gloeosporioides s. s. (ZYH-1-3-2-Cg1) caused mild symptoms on kiwifruit and persimmon at the wounded sites, and C. nymphaeae (TSK-1-Cg2) caused mild symptoms on kiwifruit, satsuma mandarin, and persimmon at the wounded sites. These results indicated that all seven Colletotrichum species have a wide host range, with C. fructicola and C. siamense appearing to be more aggressive than the other species.

In the present study, seven Colletotrichum species from three species complexes were identified in loquat in Japan based on a combination of their morphological characteristics and multiple gene phylogenetic analysis: C. fioriniae and C. nymphaeae from the C. acutatum species complex; C. karsti from the C. boninense species complex; and C. aenigma, C. fructicola, C. gloeosporioides s. s., and C. siamense from the C. gloeosporioides species complex. Of the seven species, C. fioriniae, C. nymphaeae, C. fructicola, C. gloeosporioides s. s., and C. siamense have been reported to be associated with loquat (Damm et al. 2020; Farr and Rossman 2023; James et al. 2014; Juárez‑Vázquez et al. 2019; Kuang et al. 2021; Naz et al. 2017; Sato et al. 2013). Only C. fioriniae, C. nymphaeae, and Colletotrichum sp. (MAFF 424604) have been recorded in Japan (https://www.gene.affrc.go.jp/databases-micro_search_en.php?pldis=5601). Careful checking of the ACT, GAPDH, TUB2, and ITS sequences of Colletotrichum sp. (MAFF 242604) revealed that all the sequences were similar to C. siamense. Moreover, the GAPDH and TUB2 sequences, which are crucial DNA barcodes (Cannon et al. 2012), were identical to our identified C. siamense (FuchuS-1-Cg1). Therefore, we suspected Colletotrichum sp. (MAFF 242604) was C. siamense, which led us to conclude that the three species C. fioriniae, C. nymphaeae, and C. siamense were previously found in Japan. Nevertheless, the finding in the present study is the first report of C. karsti and C. aenigma associated with loquat anthracnose and the first record of C. fructicola and C. gloeosporioides s. s. in Japan.

A pathogenicity test on the loquat fruits verified that all species produced the symptoms at the wounded sites, and C. nymphaeae, C. fructicola, and C. siamense produced slight symptoms at the intact sites. This finding indicated that wounding is conducive to the pathogenicity of these isolates. Several previous studies also showed that wounding increases the infectiousness and virulence of Colletotrichum spp. (de Aguiar Carraro et al. 2022; Guo et al. 2022; Jiang et al. 2014; Silva et al. 2020; Tan et al. 2023). Moreover, a pathogenicity test on the other plants showed that the isolates generally have the potential for cross-infectivity. However, the isolates behaved differently; for example, C. fioriniae (KMI-2-2-Ca2), C. fructicola (ZYH-3-1-2-Ca1), and C. siamense (FuchuS-3-Cg2) infected all the tested plants, while C. karsti (YMZ-2-1-Cg1) and C. aenigma (STO-2-2-2-Cg2) failed to infect satsuma mandarin and peach. According to previous reports, all the species identified in the present study have a wide host range and worldwide distribution (Damm 2012a, 2012b; Jayawardena et al. 2021; Weir et al. 2012).

Colletotrichum fioriniae has been recorded to be associated with numerous plants including kiwifruit, persimmon, peach, and satsuma mandarin (Jayawardena et al. 2021; Lee et al. 2018, 2020; Poti et al. 2023; Sato et al. 2013; Tan et al. 2022; Tashiro et al. 2018; Xu et al. 2022). Colletotrichum fructicola, C. siamense, and C. nymphaeae have also been recorded in all the tested plants in this study, excepted satsuma mandarin (Carraro et al. 2019; Chang et al. 2017; Damm et al. 2020; Evallo et al. 2022; Hassan et al. 2019; Huang et al. 2022; Kim et al. 2018; Kuang et al. 2021; James et al. 2014; Lee et al. 2020; Moreira et al. 2020; Poti et al. 2023; Tan et al. 2022). Colletotrichum aenigma has been identified in more than 20 plant species including kiwifruit and persimmon (Andrioli et al. 2021; Far and Rossman 2023; Wang et al. 2019), but it is not found in peach, loquat, and satsuma mandarin, which is consistent with the pathogenicity tests of the present study. Colletotrichum gloeosporioides has been recorded in all the plants tested in this study (Deng et al. 2017; Huang et al. 2013; Li et al. 2017; Naz et al. 2017; Poti et al. 2023; Zhang 2008). However, the isolate from this study failed to cause symptoms on satsuma mandarin and peach. Colletotrichum karsti is known to be a pathogenic pathogen in numerous plants as well as peach, kiwifruit, and persimmon (Far and Rossman 2023; Poti et al. 2023; Tan et al. 2022; Wang et al. 2015); however, there is no report in loquat and satsuma mandarin. Overall, due to cross-infectivity of all the Colletotrichum species identified in loquat to other economic plants, disease management is necessary to prevent damage to loquat production as well as other economic fruits.

In conclusion, the seven species were identified in loquat in Japan and have different cross-infection properties across other economic plants. The findings gained from this study are crucial for epidemiology and provide a cautionary tale for disease management.

Acknowledgement

We are grateful to Dennis Murphy, The United Graduate School of Agricultural Sciences, Ehime University, Japan, for editing the English of our manuscript. This work was supported by a Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan scholarship (grant number MEXT-202138).

Andrioli J, Scariot FJ, Delamare APL, Echeverrigaray S (2021) Morphological characterization and molecular identification of Colletotrichum species associated to sweet persimmon anthracnose in Southern Brazil. Cienc Rural 51. doi: 10.1590/0103-8478cr20200728
Cai L, Hyde KD, Taylor PWJ, Weir BS, Waller JM, Abang MM, Zhang JZ, Yang YL, Phoulivong S, Liu ZY, Prihastuti H, Shivas RG, McKenzie EHC, Johnston PR (2009) A polyphasic approach for studying Colletotrichum. Fungal Divers 39:183–204
Cannon PF, Damm U, Johnston PR, Weir BS (2012) Colletotrichum current status and future directions. Stud Mycol 73:181–213
Cao S, Zhen Y, Yang Z, Tang S, Jin P, Wang K, Wang X (2008) Effect of methyl jasmonate on the inhibition of Colletotrichum acutatum infection in loquat fruit and the possible mechanisms. Postharvest Biol Technol 49:301–307. https://doi.org/10.1016/j.postharvbio.2007.12.007
Cao S, Zheng Y (2010) Effect of 1-methylcyclopropene on anthracnose rot caused by Colletotrichum acutatum and disease resistance in loquat fruit. J. Sci Food Agric 90:2289–2294
Carbone I, Kohn L (1999) A method for designing primer sets for speciation studies in filamentous ascomycetes. Mycologia 91:553–556
Carraro TA, Lichtemberg PSF, Michailides TJ, Pereire WV, Figueiredo JAG, May-De Mio LL (2019) First report of Colletotrichum fructicola, C. nymphaeae, and C. melonis causing persimmon anthracnose in Brazil. Plant Dis 103:2692
Cha DS, Eun JS, Jeon H (2011) Anti-inflammatory and antinociceptive properties of the leaves of Eriobotrya japonica. J Ethnopharmacol 134:305–312
Chang T, Hassan O, Jeon JY, Shin JS, Oh N, Lim TH (2017) First report of anthracnose of persimmon (Diospyros kaki L. f.) caused by Colletotrichum siamense in Korea. Plant Dis 102:443
Corda ACI (1831) Die Pilze Deutschland. In: Sturm J (ed) Deutschlands flora in Abbildungen nach der Natur mit Beschreibungen. Sturm Nürnberg 3:33–64
Damm U, Cannon PF, Wondenberg JHC, Crous PW (2012a) The Colletotrichum acutatum species complex. Stud Mycol 73:37–113
Damm U, Cannon PF, Wondenberg JHC, Johnston PR, Weir BS, Tan YP, Shivas RG, Crous PW (2012b) The Colletotrichum boninense species complex. Stud Mycol 73:1–36
Damm U, Sun YC, Huang CJ (2020) Colletotrichum eriobotryae sp. nov. and C. nymphaeae, the anthracnose pathogens of loquat fruit in central Taiwan, and their sensitivity to azoxystrobin. Mycol Prog 19:367–380
de Aguiar Carraro T, Lichtemberg PSF, Michailides TJ, Miranda Borges MI, Pereira WV, May De Mio LL (2022) Identification and characterization of Colletotrichum species associated with anthracnose on persimmon in Brazil. Fungal Biol 126:235–249
Deng JC, Guan YM, Wu LJ, Zhang YY (2017) First report of anthracnose caused by Colletotrichum gloeosporioides on Actinidia arguta in China. Plant Dis 101:1032
Eaton MJ, Edwards S, Inocencio HA, Machado FJ, Nuckìes EM, Farman M, Gauthier NA, Vaillancourt LJ (2021) Diversity and cross-infection potential of Colletotrichum causing fruit rots in mixed-fruit orchards in Kentucky. Plant Dis 105:1115–1128
Evallo E, Taguiam JD, Balendres MA (2022) Colletotrichum fructicola associated with fruit anthracnose of persimmon. J Phytopathol 170:194–201
Far DF, Rossman AY (2023) Fungal Databases, U.S. National Fungus Collections, ARS, USDA. Available via DIALOG. https://nt.ars-grin.gov/fungaldatabases/. Accessed 25 Jan 2023
Glass NL, Donaldson GC (1995) Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes. Appl Environ Microbiol 61:1323–1330
Guo Z, Luo CX, Wu HJ, Peng B, Kang BS, Liu LM, Zhang M, Gu QS (2022) Colletotrichum species associated with anthracnose disease of watermelon (Citrullus lanatus) in China. J Fungi 8:790
Hassan O, Lee DW, Chang T (2019) First report of anthracnose of persimmon caused by Colletotrichum nymphaeae in Korea. Plant Dis 103:1772
Huang F, Chen GQ, Hou X, Fu YS, Cai L, Hyde KD, Li HY (2013) Colletotrichum species associated with cultivated citrus in China. Fungal Divers 61:61–74
Huang L, Sheng J, Song W, Zheng D, Song S, Xu X, Yu J, Liu Q, Liu Y, Tang W (2022) First report of leaf spot caused by Colletotrichum fructicola on kiwi in China. Plant Dis 106:2760
Hyde KD, Nilsson RH, Alias SA, Ariyawansa HA, Blair JE, Cai L, de Cock AWAM, Dissanayake AJ, Glockling SL, Goonasekara ID, Gorczak M, Hahn M, Jayawardena RS, van Kan JAL, et al. (2014) One stop shop: backbones trees for important phytopathogenic genera: I. Fungal Divers 67:21–125
Hyde KD, Norphanphoun C, Maharachchikumbura SSN, Bhat DJ, Jones EBG, Bundhun D, Chen YJ, Bao DF, Boonmee S, Calabon MS, Chaiwan N, Chethana KWT, Dai DQ, Dayarathne MC, et al. (2020) Refined families of Sordariomycetes. Mycosphere 11:305–1059
Hyde KD, Tennakoon DS, Jeewon R, Bhat DJ, Maharachchikumbura SSN, Rossi W, Leonardi M, Lee HB, Mun HY, Houbraken J, Nguyen TTT, Jeon SJ, Frisvad JC, Wanasinghe DN, et al. (2016) Fungal diversity notes 1036–1150: taxonomic and phylogenetic contributions on genera and species of fungal taxa. Fungal Divers 96:1–242
James RS, Ray J, Tan YP, Shivas RG (2014) Colletotrichum siamense, C. theobromicola and C. queenslandicum from several plant species and the identification of C. asianum in the Northern Territory, Australia. Australasian Plant Dis Notes 9:138
Jayawardena RS, Bhunjun CS, Hyde KD, Gentekaki E, Itthayakorn P (2021) Colletotrichum: lifestyles, biology, morpho-species, species complexes and accepted species. Mycosphere 12:519–669
Jayawardena RS, Hyde KD, Damm U, Cai L, Liu M, Li XH, Zhang W, Zhao WS, Yan JY (2016) Notes on currently accepted species of Colletotrichum. Mycosphere 7:1195–1260
Jiang J, Zhai H, Li H, Wang Z, Chen Y, Hong N, Wang G, Chofong GN, Xu W (2014) Identification and characterization of Colletotrichum fructicola causing black spots on young fruits related to bitter rot of pear (Pyrus bretschneideri Rehd.) in China. Crop Prot 58:41–48
Juárez‑Vázquez SB, Silva-Rojas HV, Rebollar-Alviter A, Maidana-Ojeda M, Osnaya-González M, Fuentes‑Aragón D (2019) Phylogenetic and morphological identification of Colletotrichum godetiae, a novel pathogen causing anthracnose on loquat fruits (Eriobotrya japonica). J Plant Dis Prot 126:593–598
Katoh K, Rozewicki J, Yamada KD (2019) MAFFT online service: multiple sequence alignment, interactive sequence choice and visualization. Brief Bioinform 20(4):1160–1166
Khouya T, Ramchoun M, Elbouny H, Hmidani A, Bouhlali ET, Alem C (2022) Loquat (Eriobotrya japonica (Thunb) Lindl.): evaluation of nutritional value, polyphenol composition, antidiabetic effect, and toxicity of leaf aqueous extract. J Ethnopharmacol 209:115473
Kim GH, Choi DH, Park SY, Koh YJ (2018) First report of anthracnose caused by Colletotrichum nymphaeae on kiwiberry in Korea. Plant Dis 102:1455
Kuang W, Cui R, Xiang M, Liu B, Wang Z, Guo D, Ma J (2021) First report of Colletotrichum fructicola causing anthracnose on loquat in Jiangxi province, China. J Plant Pathol 103:1053–1054
Kuraoka-Oliveira ÂM, Radai JAS, Lietão MM, Cardoso CAL, Silva-Filho SE, Kassuya CAL (2020) Anti-inflammatory and anti-arthritic activity in extract from the leaves of Eriobotrya japonica. J Ethnopharmacol 249:112418
Lee D, Hassan O, Chang T (2018) First report of peach (Prunus persica) anthracnose caused by Colletotrichum fioriniae in Korea. Plant Dis 102:2650
Lee DM, Hassan O, Chang T (2020) Identification, characterization, and pathogenicity of Colletotrichum species causing anthracnose of peach in Korea. Microbiology 48:210–218
Lee SB, Milgroom MG, Taylor JW (1988) A rapid, high yield mini-prep method for isolation of total genomic DNA from fungi. Fungal Genet Rep 35. doi: 10.4148/1941-4765.1531
Li L, Pan H, Chen MY, Zhang SJ, Zong CH (2017) First report of anthracnose caused by Colletotrichum gloeosporioides on kiwifruit (Actinidia chinensis) in China. Plant Dis 101:2151
Li X, Xu C, Chen K (2016). Nutritional and composition of fruit cultivars: loquat (Eriobotrya japonica Lindl.). In: Simmonds MSJ, Preedy VR (eds) Nutritional composition of fruit cultivars. Academic Press, US, pp. 371–394. https://doi.org/10.1016/B978-0-12-408117-8.00016-7
Liu AY, Chen WX, Gu H, Shi JY, Li J (2007) Biological characteristic of pathogenic fungus causing anthracnose of loquat fruit. Acta Hortic 750:465–470
Maharachchikumbura SN, Hyde KD, Jones EBG, McKenzie EHC, McKenzie EHC, et al. (2016) Families of Sordariomycetes. Fungal Divers 79:1–371
Miller MA, Pfeiffer W, Schwartz T (2010) Creating the CIPRES Science Gateway for inference of large phylogenetic trees. In: 2010 Gateway Computing Environments Workshop (GCE), New Orleans, LA, USA, pp. 1–7
Moral J, Agustí-Brisach C, Raya MC, Jurado-Bello J, López-Moral A, Roca LF, Chattaoui M, Rhouma A, Nigro F, Sergeeva V, Trapero A (2021) Diversity of Colletotrichum species associated with olive anthracnose worldwide. J Fungi 7:741
Moreira RR, Silva GA, De Mio LLM (2020) Colletotrichum acutatum complex causing anthracnose on peach in Brazil. Austral Plant Pathol 49:210–218
Naz F, Abbas MF, Rauf CA, Tariq A, Mumtaz A, Irshad G, Shaheen FA, Hassan I (2017) First report of Colletotrichum gloeosporioides causing anthracnose on loquat in Pakistan. Plant Dis 101:1150
O’Donnell K, Cigelnik E (1997) Two divergent intragenomic rDNA ITS2 types within a monophyletic lineage of the fungus Fusarium are nonorthologous. Mol Phylogenet Evol 7:103–116
Palou L, Sánchez-Torres P, Nontesinos-Herrero C, Taverner V (2016) Incidence and etiology of postharvest fungal diseases of loquat fruit (Eriobotrya japonica (Thunb.) Lindl. cv. ‘Algerie’) in Alacant province (Spain). Eur J Plant Pathol 146:847–860. https://doi.org/10.1007/s10658-016-0964-0
Poti T, Kisaki G, Arita K, Akimitsu K (2023) Identification and characterization of Colletotrichum species causing kiwifruit anthracnose in Kagawa Prefecture, Japan. J Gen Plant Pathol 89:84–90
Sato T, Moriwaki J, Misawa T (2013) Molecular re-identification of strains of the Colletotrichum acutatum species complex deposited in the NIAS Genebank and morphological characteristics of its member species. JARQ 47:295–305
Shen Y, Chen C, Cai N, Yang R, Chen J, Kahramanoǧlu İ, Okatan V, Rengasamy KRR, Wan C (2021) The antifungal activity of loquat (Eriobotrya japonica Lindl.) leaves extract against Penicillium digitatum. Front Nutr 8:663584
Silva DDD, Ades PK, Taylor PWJ (2020) Pathogenicity of Colletotrichum species causing anthracnose of Capsicum in Asia. Plant Pathol 70:875–884
Talhinhas P, Baroncelli R (2021) Colletotrichum species and complexes: geographic distribution, host range and conservation status. Fungal Divers 110:109–198
Tan Q, Schnabel G, Chaisiri C, Yin LF, Yin WX, Luo CX (2022) Colletotrichum species associated with peaches in China. J Fungi 8:313
Tan X, Zhao J, Qiao G, Liu J, Hsiang T, Yu Z, Qin W (2023) Morphological characterization and pathogenicity of Colletotrichum aenigma and C. siamense causing anthracnose on Euonymus japonicus in Beijing, China. Plant Pathol 73:430–441
Tashiro N, Urakawa A, Shoji K, Matsuo Y, Sato T (2018) Postharvest anthracnose on Satsuma mandarin orange caused by Colletotrichum fioriniae. J Gen Plant Pathol 84:381–385
Templeton MD, Rikkerink EH, Solon SL, Crowhurst RN (1992) Cloning and molecular characterization of the glyceraldehyde-3-phosphate dehydrogenase-encoding gene and cDNA from the plant pathogenic fungus Glomerella cingulata. Gene 122:225–230
Tian S, Qin G, Li B (2011) Loquat (Eriobotrya japonica L.). In: Yahai MY (ed) Postharvest biology and technology of tropical and subtropical fruits. Woodhead Publishing, UK, pp. 424–442. https://doi.org/10.1533/9780857092885.424
Tian SP, Li BQ, Ding ZS (2007) Physiological properties and storage technologies of loquat fruit. In: Jaime A, Teixeira da S (eds) Fresh produce, Global Science Books, Isleworth, UK, pp. 76–81
Wang J, Ai CX, Yu XM, An M, Sun S, Gao R (2015) First report of Colletotrichum karsti causing anthracnose on persimmon leaves in China. Plant Dis 100:532
Wang Y, Qin HY, Liu YX, Fan ST, Sun D, Yang YM, Li CY, Ai J (2019) First report of anthracnose caused by Colletotrichum aenigma on Actinidia arguta in China. Plant Dis 103:372
Weir BS, Johnston PR, Damm U (2012) The Colletotrichum gloeosporioides species complex. Stud Mycol 73:115–180
White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis M, Gelfand D, Sninsky J, White T (eds) PCR protocols: a guide to methods and applications. Academic Press, San Diego, CA, pp. 315–322
Xu X, Zheng D, Lan J, Song W, Song S, Huang L, Liu Y, Tang W (2022) First report of post-harvest anthracnose of kiwifruit caused by Colletotrichum fioriniae in Liaoning and Sichuan Province, China. Plant Dis. doi: 10.1094/PDIS-07-22-1673-PDN
Yu L, Lyu C, Tang Y, Lan G, Li Z, She X, He Z (2022) Anthracnose: a new leaf disease on Radermachera sinica (China Doll) in China. Plant Dis 106:2034–2309
Zhang JZ (2008) Anthracnose of persimmon caused by Colletotrichum gloeosporioides in China. Asian Australas J Plant Sci Biotechnol 2:50–54
Zhang YJ, Zhang S, Liu XZ, Wen HA, Wang M (2010) A simple method of genomic DNA extraction suitable for analysis of bulk fungal strain. Lett Appl Microbiol 51:114–118

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Colletotrichum species associated with loquat anthracnose in Kagawa and Tokushima prefectures, Japan

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Abstract

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Introduction

Materials and methods

Sample collection, fungal isolation, and morphological examination

DNA extraction, PCR amplification, and sequencing

Phylogenetic analysis

Pathogenicity test

Results

Fungal isolation and morphological characterization

Phylogenetic analysis

Pathogenicity on loquat fruit and cross-infection on the other fruits

Discussion

Declarations

Acknowledgement

References

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