Short Communication
Taxonomic re-examination of Japanese isolates of the genus Lasiodiplodia
2023 Volume 64 Issue 2 Pages 74-82
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2023 Volume 64 Issue 2 Pages 74-82
Lasiodiplodia species cause dieback, shoot blight, fruit rot, and stem-end rot of woody plants. Recently, studies on taxonomic revision of Lasiodiplodia species have been conducted globally and have led to more clarification of the species diversity of this genus. However, only three species of the genus have been reported in Japan. To evaluate the species diversity of Lasiodiplodia in Japan, we re-examined the taxonomical positions of 30 isolates based on their morphological and cultural characteristics and phylogenetic relationships. Phylogenetic analyses were performed using a matrix comprising ITS, tef1, tub2, and rpb2 sequences, and the results indicate that the examined Japanese isolates belong to six species: Lasiodiplodia brasiliensis, L. hormozganensis, L. pseudotheobromae, L. thailandica, L. theobromae, and Lasiodiplodia sp. Moreover, three of these species were newly added to the Japanese mycoflora.
The genus Lasiodiplodia (Botryosphaeriaceae) was established by Ellis and Everhart in 1896 (Clendenin, 1896). This genus comprises several plant pathogenic fungi associated with fruit rot, dieback, and shoot blights in fruit trees, mainly in the subtropical and tropical regions (Phillips et al., 2013). Those elliptical or long ellipsoidal conidia with a single septum are characterized by light-brown to dark-brown coloration and often show longitudinal striations after maturation (Phillips et al., 2013; de Silva, Phillips, Liu, Lumyong, & Hyde, 2019). Crous et al. (2006) evaluated the phylogeny of Botryosphaeriaceae using large-subunit rDNA sequences, and the results indicated the existence of cryptic genera and species within hitherto known taxa. Subsequently, many other cryptic species of the genus Lasiodiplodia have been discovered from the known host plants (Burgess et al., 2006; Alves, Crous, Correia, & Phillips, 2008; Abdollahzadeh, Javadi, Goltapeh, Zare, & Phillips, 2010).
The Lasiodiplodia theobromae (Pat.) Griffon & Maubl. species complex includes numerous cryptic and potential species (Ismail et al., 2012). Previous molecular phylogenetic studies on this complex used the internal transcribed spacer (ITS) and translation elongation factor 1-alpha (tef1) regional sequences; however, they could not indicate the species limit within the complex but they did indicate the interspecies limit for among other Lasiodiplodia species (Alves et al., 2008; Cruywagen, Slippers, Roux, & Wingfield, 2017; Slippers, Crous, Jami, Groenewald, & Wingfield, 2017). Subsequently, molecular phylogenetic analysis using the combined data matrix, comprising the three DNA regions of ITS, tef1, and beta tubulin (tub2), revealed the species limit of Lasiodiplodia species, including that for the L. theobromae species complex (Phillips, Hyde, Alves, & Liu, 2019; Jayasiri et al., 2019). Phylogenetic studies have also revealed that several Lasiodiplodia species inhabit specific host plants. For example, four Lasiodiplodia species were isolated from Magnolia candollei (Blume) H. Keng (de Silva et al., 2019), and 12 taxa were isolated from Aquilaria crassna Pierre ex Lecomte (Wang et al., 2019) in Laos. Conversely, many species have been described by the overestimation of sequence differences, although they still lack morphological and ecological information. Zhang et al. (2021) analyzed the phylogeny of 34 Lasiodiplodia species using a combined matrix of ITS, tef1, tub2, and DNA-directed RNA polymerase II second largest subunit (rpb2) regional sequences and concluded that the morphologically similar species described by only phylogeny- or morphology-based criteria should be synonymized under known species, such as L. theobromae.
In Japan, L. theobromae had been reported from plants of 33 genera of 23 families (the database of Plant Disease in Japan, https://www.gene.affrc.go.jp/databases-micro_pl_diseases.php; July 2020). In the last decade, L. parva A.J.L. Phillips, A. Alves & Crous, and L. pseudotheobromae A.J.L. Phillips, A. Alves & Crous were reported to be found on the remote southern islands of Japan, Ogasawara Islands and Miyako Islands. They were identified using a combined matrix of rDNA ITS and 28S rDNA regions and based on those morphological characteristics (Sato, Uzuhashi, Hosoya, & Hosaka, 2010; Kageyama, 2010; Sato, Ono, Tanaka, & Hattori, 2016; Tanaka et al., 2017). However, the number of Lasiodiplodia species reported from Japan might be too small compared with the richness of the host plant diversity. In the case of Pseudocercospora species, plant pathogenic fungi having a relatively limited host range, and the fungal species diversity is subjected to the host plant diversity (Nakashima, Motohashi, Chen, Groenewald, & Crous, 2016; Chen et al., 2022). And several plant pathogenic species of the genus Phyllosticta have a relatively limited host range, and the species diversity relies on the host plant diversity (Motohashi, Inaba, Anzai, Takamatsu, & Nakashima, 2009; Wikee et al. 2013; Hattori, Motohashi, Tanaka, & Nakashima, 2020). Similarly, comprehensive taxonomical studies on Lasiodiplodia species based on the integrated species concept along with multilocus phylogenetic analyses and detailed morphological observation could reveal the diversity of fungal species that are strongly related to the diversity of host plants in this geographical area. In addition, economically important crops are often infected by Lasiodiplodia species during the cultivation or postharvest stages. For accurate and rapid diagnoses of the consequent diseases, information on species diversity through molecular phylogeny, morphology, and cultural characteristics is required in the field and for plant quarantine. This study re-examined the Japanese isolates of Lasiodiplodia species obtained from various host plants to evaluate the taxonomic position with the modern species criteria using phylogenetic and morphological analyses.
A total of 30 isolates were investigated, including 23 isolates of the genus Lasiodiplodia deposited in the Microbiological Genebank, National Institute of Agrobiological Science (MAFF); 5 newly established isolates from the symptomatic twigs, fruits, and leaves of Mangifera indica L. collected from Miyako Island, Japan; and 2 isolates from the symptomatic fruits of M. indica imported from the Philippines (Table 1). All isolates were incubated on a potato dextrose agar medium (PDA; Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) or malt agar medium (MA; Becton Dickinson, MD, USA) at 25 °C under light diffusion. Additionally, newly established isolates were stored and maintained at the Culture Collection of the Mycological Herbarium, Mie University (TSU-MUCC), Tsu, Mie, Japan.
| Fungal species | MUCC | MAFF | Host Family | Host | Locality | Identified in previous study | Reference |
| Lasiodiplodia brasiliensis | 2553 | − | Anacardiaceae | Mangifera indica | Tokyo a | − | This study |
| L. brasiliensis | 2725 | 241241 | Caricaceae | Carica papaya | Ibaraki | L. theobromae | − |
| L. brasiliensis | 2729 | 242316 | Anacardiaceae | M. indica | Tokyo, Hahajima Is. | Lasiodiplodia sp. | − |
| L. brasiliensis | 2740 | 306028 | Annonaceae | Annona squamosa | Tokyo, Chichijima Is. | L. theobromae | Sato et al., 2010 |
| L. hormozganensis | 2723 | 239129 | Pandanaceae | Pandanus odoratissimus | Kagoshima, Yoron Is. | L. theobromae | Yumiki et al., 2008 |
| L. hormozganensis | 2724 | 240591 | Xanthorrhoeaceae | Aloe vera | Okinawa, Miyako Is. | L. theobromae | Kageyama, 2010 |
| L. hormozganensis | 2732 | 243701 | Asparagaceae | Agave sisalana | Tokyo, Hahajima Is. | L. parva | − |
| L. hormozganensis | 2733 | 243706 | Cannabaceae | Trema orientalis | Tokyo, Hahajima Is. | L. parva | Sato et al., 2016 |
| L. hormozganensis | 2734 | 243707 | Araceae | Monstera sp. | Tokyo, Hahajima Is. | L. parva | − |
| L. hormozganensis | 2737 | 244451 | Malvaceae | Hibiscus rosa-sinensis | Tokyo, Hahajima Is. | L. parva | − |
| L. hormozganensis | 2741 | 306514 | Nymphalidae | Idea leuconoe (insects) | Okinawa, Okinawa Is. | L. theobromae | Nago & Matsumoto, 1994 |
| L. hormozganensis | 2742 | 306515 | Hernandiaceae | Hernandia nymphaeifolia | Okinawa, Ishigaki Is. | L. theobromae | Nago & Matsumoto, 1994 |
| L. hormozganensis | 2743 | 306516 | Apocynaceae | Parsonsia alboflavescens | Okinawa, Ishigaki Is. | L. theobromae | Nago & Matsumoto, 1994 |
| L. hormozganensis | 2883 | 237946 | Arecaceae | Cocos nucifera | Okinawa, Miyako Is. | L. theobromae | − |
| L. pseudotheobromae | 2726 | 241277 | Rasaceae | Rosa sp. | Wakayama | L. theobromae | − |
| L. thailandica | 2738 | 244514 | Crassulaceae | Bryophyllum pinnatum | Tokyo, Chichijima Is. | L. pseudotheobromae | Tanaka et al., 2017 |
| L. thailandica | 2587 | − | Anacardiaceae | M. indica | Okinawa, Miyako Is. | − | This study |
| L. theobromae | 2577 | − | Anacardiaceae | M. indica | Okinawa, Miyako Is. | − | This study |
| L. theobromae | 2590 | − | Anacardiaceae | M. indica | Okinawa, Miyako Is. | − | This study |
| L. theobromae | 2604 | − | Anacardiaceae | M. indica | Tokyo a | − | This study |
| L. theobromae | 2632 | − | Anacardiaceae | M. indica | Okinawa, Miyako Is. | − | This study |
| L. theobromae | 2746 | 243205 | Malvaceae | Theobroma cacao | Tokyo, Chichijima Is. | L. theobromae | − |
| Lasiodiplodia sp. | 2739 | 306027 | Rutaceae | Citrus sinensis | Tokyo, Chichijima Is. | L. theobromae | Sato et al., 2010 |
| Lasiodiplodia sp. | 2728 | 241893 | Anacardiaceae | Mangifera indica | Tokyo, Chichijima Is. | L. pseudotheobromae | Sato et al., 2010 |
| Lasiodiplodia sp. | 2735 | 243711 | Asteraceae | Wollastonia dentata | Tokyo, Chichijima Is. | L. pseudotheobromae | Sato et al., 2016 |
| Lasiodiplodia sp. | 2736 | 244435 | Amaryllidaceae | Crinum asiaticum | Tokyo, Chichijima Is. | L. pseudotheobromae | Sato et al., 2016 |
| Lasiodiplodia sp. | 2730 | 242320 | Passifloraceae | Passiflora edulis | Tokyo, Chichijima Is. | L. theobromae | − |
| Lasiodiplodia sp. | 2731 | 242322 | Arecaceae | Phoenix roebelenii | Tokyo, Hachijojima Is. | L. theobromae | − |
| Lasiodiplodia sp. | 2744 | 306517 | Apocynaceae | Anodendron affine | Okinawa, Okinawa Is. | L. theobromae | Nago & Matsumoto 1994 |
| Lasiodiplodia sp. | 2745 | 411002 | Fabaceae | Laburnum anagyroides | Tochigi | L. theobromae | − |
a Symptomatic fruits imported from the Philippines were intercepted at Tokyo.
The fungal structures grown on media were observed under a compound microscope (Axio Imager A1, Zeiss, Göttingen, Germany). Subsequently, the isolates were transferred onto pine needle agar medium (PNA; Crous, Verkleij, Groenewald, & Houbraken, 2019) and incubated at 25 °C under light diffusion for 2 mo. This was performed to induce the formation of the conidiomata and conidia. The length and width of mature conidia were measured, and the length/width ratio (L/W) was calculated. The optimum growth temperature on PDA at 25 °C, 30 °C, and 35 °C and the colony growth rate on a PDA at 25 °C under dark conditions were examined. After 48 h, the major and minor diameters of the mycelial colonies were measured. The initial size of the mycelial disk was subtracted from the final size of the colony.
Genomic DNA was extracted from the mycelial colonies after 14 d of inoculation on PDA plates using the DNeasy UltraClean Microbial Kit (QIAGEN, Hilden, Germany) as per the manufacturer’s instructions. Targeted sequences of ITS and partial sequences of tef1, tub2, and rpb2 regions were amplified. The PCR and sequencing analysis conditions were the same as those used by Hattori, Ando, and Nakashima (2021). The obtained sequences were further assembled, concatenated, and aligned with 82 sequences from the genus Lasiodiplodia retrieved from the DNA Data Bank of Japan using MEGA v. 5.2 software (Tamura et al., 2011). The aligned matrix was realigned with the matrix published by Zhang et al. (2021) as a reference alignment using MAFFT v.7 (Katoh, Rozewicki, & Yamada, 2019). Maximum likelihood (ML) and Bayesian inference (BI) analyses were used to estimate the phylogenetic relationships. ML analyses were conducted using RAxML-NG v. 1.0.2 (Kozlov, Darriba, Flouri, Morel, & Stamatakis, 2019) with the nucleotide substitution model selected by ModelTest-NG v. 0.1.6 (Darriba et al., 2020). The strength of the internal branches from the resultant trees was tested further using bootstrap analysis (Felsenstein, 1985), which involved 500 replications. BI analyses were conducted using Mr. Bayes 3.2.6 (Ronquist & Huelsenbeck, 2003) to estimate tree topologies’ posterior probabilities (PPs). MCMC analysis was conducted for 10,000,000 generations, and trees were sampled and saved every 1,000 generations. The first 30% of saved trees were discarded as “burn-in” based on the effective sample size (ESS) calculated by Tracer v. 1.7.1 (Rambaut, Drummond, Xie, Baele, & Suchard, 2018). The representative sequences for all taxa were uploaded to GenBank (Supplementary Table S1), and their sequence alignments were deposited in TreeBASE number S29484. As the results, the ITS (387) + tef1 (262) + tub2 (422) + rpb2 (525) concatenated data matrix consisting of 1,596 characters (including gaps) was generated. The evolutional models were selected and tagged with K80+I for ITS, HKY+G4 for tef1, TrN+I for tub2, and TN93ef+G4 for rpb2 for ML analysis and HKY+I+G for BI analysis. Japanese Lasiodiplodia isolates examined in this study were divided into six clades: L. brasiliensis, L. hormozganensis, L. pseudotheobromae, L. thailandica, L. theobromae, and a phylogenetic group of Lasiodiplodia (Lasiodiplodia sp. 1).
Lasiodiplodia brasiliensis M.S.B. Netto, M.W. Marques & A.J.L. Phillips, Fungal Diversity 67: 134, 2014. Supplementary Fig. S1.
Conidiomata formed on PNA within 21−35 d, pycnidial, globose to oblate, thick-walled, uniloculate, light gray to dark gray, covered with hyphae; paraphyses hyaline, cylindrical, septate, rounded at the apex; conidiogenous cells hyaline, cylindrical, sporulating holoblastically, 6.5-28 × 2-6 µm (n = 17). Conidia oblong or rarely ellipsoidal, rounded at both ends, initially hyaline to light brown, with granular content, aseptate, becoming light brown to dark brown and one-septate with longitudinal striations on conidiogenous cells, 20-27 × 11-15 µm, L/W= 1.79 (n = 119, MUCC 2740).
Cultural Characteristics: on PDA: colonies with dense aerial mycelia, light gray in color, olive to dark gray at the center, white to light gray around the edge, reaching 90 mm at 7 d after inoculation (MUCC 2553, MUCC 2725), growing in the range 20-35 °C. The optimum growth temperature was in the range of 25-35 °C.
Isolates Examined: Carica papaya - on fruits, Japan, Ibaraki, Tsukuba, Dec 2008, by Y. Otani (MAFF 241241 = MUCC 2725); Annona squamosa, Japan, Tokyo, Ogasawara, Chichijima Island, 1986, by T. Sato (MAFF 306028 = MUCC 2740); Mangifera indica - on twigs, Japan, Tokyo, Ogasawara, Hahajima Island, Aug 2002, by T. Ono (MAFF 242316 = MUCC 2729); on fruits imported from the Philippines, intercepted at Japan, Tokyo, Chiyoda, 2 Jul 2018, by Y. Hattori (MUCC 2553).
Note: Four of the examined isolates were located in the same clade as an ex-type isolate of L. brasiliensis (CMM 4015) (Fig. 1). Although the morphological characteristics of MUCC 2740 were almost identical to those of MUCC 2553, MUCC 2725 and MUCC 2729, the former is slightly far from the main clade. The conidia size of these three isolates was slightly smaller than that observed in the previous study (22.7-29.2 × 11.7-17.0 µm in Netto et al., 2014), whereas the L/W ratio was almost the same (1.8 in Netto et al., 2014). The optimum growth temperature was ca. 32 °C in the study by Netto et al. (2014). In contrast, the optimum growth temperature ranged from 30 °C to 35 °C in this study. Lasiodiplodia brasiliensis has been reportedly found in 11 host plant species. These host plant species are distributed across Mexico, Puerto Rico, Brasil, Laos, Thailand, Malaysia, and Australia (Farr & Rossman, 2022). This is the first report indicating the species’ presence in Japan and a new host plant, C. papaya.
Lasiodiplodia hormozganensis Abdollahzadeh, Zare & A.J.L. Phillips, Persoonia 25: 6, 2010. Supplementary Fig. S2.
Conidiomata formed on PNA within 13-52 d, pycnidial, globose to oblate, thick-walled, uniloculate, light brown to dark gray, covered with hyphae; paraphyses hyaline, rounded at the apex, aseptate; conidiogenous cells hyaline, cylindrical, sporulating holoblastically, 6-15 × 1.7-8 µm (n = 23). Conidia ellipsoidal or rarely oblong, rounded at both ends, initially hyaline, with granular content, aseptate, becoming light brown to dark brown and one-septate after releasing from the conidiogenous cells, with longitudinal striations, 17.5-23.5 × 10.5-13 µm, L/W= 1.73 (n = 100, MUCC 2724).
Cultural Characteristics: on PDA: colonies with dense aerial mycelia, dark gray to dark olive at the center, light gray to white around the edge, reaching 90 mm after 7 d of inoculation and growing in the range 20 °C-35 °C. The optimum growth temperature varies in the range 20 °C-30 °C (MUCC 2737), 25 °C-35 °C (MUCC 2723, MUCC 2724), 30 °C (MUCC 2742), 35 °C (MUCC 2883, MUCC 2733, MUCC 2732), and 30 °C-35 °C (MUCC 2734).
Isolates Examined: Pandanus odoratissimus - Japan, Kagoshima, Yoron Island, Nov, 2001, by T. Kobayashi (MAFF 239129 = MUCC 2723); Hernandia nymphaeifolia - Japan, Okinawa, Ishigaki Island, Feb 1993, by H. Nago (MAFF 306515 = MUCC 2742); Idea leuconoe - Japan, Okinawa, Okinawa Island, Oct 1991, by H. Nago (MAFF 306514 = MUCC 2741); dried culture of MUCC 2883 isolated from Cocos nucifera, Japan, Okinawa, Miyako Island, Nov 1991, by T. Kobayashi, culture MAFF 237946 (MUCC 2883); Aloe vera - Japan, Okinawa, Miyako Island, Sep 2007, by Y. Hirooka (MAFF 240591 = MUCC 2724); Parsonsia alboflavescens - Japan, Okinawa, Ishigaki Island, Feb 1993, by H. Nago (MAFF 306516 = MUCC 2743); Agave sisalana - Japan, Tokyo, Ogasawara, Hahajima Island, Sep 2012, by T. Sato (MAFF 243701 = MUCC 2732); Hibiscus rosa-sinensis - Japan, Tokyo, Ogasawara, Hahajima Island, Sep 2012, by T. Sato (MAFF 244451 = MUCC 2737); Monstera sp. - Japan, Tokyo, Ogasawara, Hahajima Island, Aug 2002, by T. Sato (MAFF 243707 = MUCC 2734); Trema orientalis - Japan, Tokyo, Ogasawara, Hahajima Island, Sep 2012, by T. Sato (MAFF 243706 = MUCC 2733).
Note: Molecular phylogenetic analyses indicated that 10 Japanese isolates and the ex-type culture of L. hormozganensis (CBS 124709) formed an independent clade (ML-BS: 62% and BI-PP: 0.96) (Fig. 1). This study is the first report of L. hormozganensis from Japan and the new host, Al. vera, Ag. sisalana, C. nucifera, He. nymphaeifolia, Hi. rosa-sinensis, I. leuconoe, Monstera sp., Pan. odoratissimus, Par. alboflavescens, and T. orientalis.
Lasiodiplodia pseudotheobromae A.J.L. Phillips, A. Alves & Crous, Fungal Diversity 28: 8, 2008.
Cultural Characteristics: on PDA: colonies with dense aerial mycelia, white, reaching 90 mm at 7 d after inoculation (MUCC 2726) and growing in the range of 20 °C-35 °C. The optimum growth temperature was 35 °C.
Isolate Examined: Rosa sp. - Japan, Wakayama, Sep 2008, by Y. Otani (MAFF 241277 = MUCC 2726).
Note: On observing the cultural characteristics on PNA, only light to dark brown mycelium covered the pine leaves at the 39 d, and conidiomata formation could not be observed. The examined isolate MUCC 2726 was located in the L. pseudotheobromae clade (ML-BS: 67%), including the ex-type isolate of L. pseudotheobromae (CBS 116459) on the resultant tree (Fig. 1). From the phylogenetic position, this examined isolate was identified as L. pseudotheobromae. According to Alves et al. (2008), the isolate of L. pseudotheobromae produced a pink pigment on PDA at 35 °C. However, MUCC 2726 did not produce the pigmentation on the medium in the same conditions. Lasiodiplodia pseudotheobromae has been isolated from various hosts globally and has also been reported from the plant genus Rosa in the Netherlands and Australia (Alves et al., 2008; Alves et al., 2019).
Lasiodiplodia thailandica T. Trakunyingcharoen, L. Lombard & Crous, Persoonia 34: 95, 2015. Supplementary Fig. S3.
Conidiomata formed on PNA within 39 d, pycnidial, aggregated, globose to subglobose, uniloculate, dark brown, thick-walled, covered with hyphae; paraphyses hyaline, rounded at the apex, aseptate; conidiogenous cells hyaline, cylindrical or rod shape, sporulating holoblastically, 4.5-15 × 1.8-4.5 µm (n = 16). Conidia ellipsoidal, rounded at both ends, initially hyaline, with granular content, aseptate, becoming light brown to dark brown and one-septate after releasing from the conidiogenous cells, with longitudinal striations, 19.5-28 × 12-16 µm, L/W = 1.71 (n = 40, MUCC 2738).
Cultural characteristics: on PDA: colonies with dense aerial mycelium, white to light gray. Colonies reaching 90 mm diameter at 7 d after inoculation and growing in the range 20 °C-35 °C. Optimum growth was observed at temperatures in the range of 25 °C-30 °C.
Isolates examined: Mangifera indica - on fruit, Japan, Okinawa, Miyako Island, Jul 2018, by Y. Hattori (MUCC 2587); Kalanchoe pinnata - Japan, Tokyo, Ogasawara, Chichijima Island, Sep 2012, by K. Tanaka (MAFF 244514 = MUCC 2738).
Note: On the resultant phylogenetic tree, MUCC 2587 and MUCC 2738 were located in the same clade as the ex-type isolate of L. thailandica (CBS 138760) (Fig. 1). Morphologically, the size of matured conidia of MUCC 2738 was within the range of that of L. thailandica. This species has been reported from woody plants only, including M. indica and Phyllanthus acidus in Thailand and Podocarpus macrophyllus and Albizia chinensis in China (Trakunyingcharoen et al., 2015; Dou, He, & Zhang, 2017; de Silva et al., 2019). This study is the first record of the herbaceous host plant for L. thailandica, and the species has been newly added to the Japanese mycoflora.
Lasiodiplodia theobromae (Pat.) Griffon & Maubl., Bulletin de la Société Mycologique de France 25: 57, 1909.
Conidiomata formed on PNA within 12−64 d, pycnidial, uniloculate, light gray to dark gray, covered with hyphae; conidiogenous cells hyaline, cylindrical or rod shape, holoblastic, 9-16.5 × 2.7-5.5 µm (n = 13). Conidia ellipsoidal or ovoid, rounded at both ends, initially hyaline, aseptate or rarely one-septate, becoming dark brown and one-septate after release from the conidiomata, with longitudinal striations, 19-29 × 12-17.5 µm, L/W = 1.71 (n = 100, MUCC 2632).
Cultural Characteristics: on PDA: colonies with dense aerial mycelia, light-dark gray, light gray, olive-buff, or dark gray at the center, white around the edge, or white at the center and light brown around the edge, reaching 90 mm at 7 d after inoculation, growing in the range 20 °C-35 °C. The optimum growth temperature varied between 30 °C (MUCC 2632 and MUCC 2746) and 30 °C-35 °C (MUCC 2577).
Isolates Examined: Mangifera indica - on fruits, Japan, Okinawa, Miyako Island, Jul 31, 2018, by Y. Hattori (MUCC 2577); on stem, Japan, Okinawa, Miyako Island, Aug 2, 2018, by Y. Hattori (MUCC 2590); on branch, Japan, Okinawa, Miyako Island, Oct 23, 2018, by Y. Hattori (MUCC 2632); on fruits imported from the Philippines, intercepted at Japan, Tokyo, Chiyoda, May 16, 2018, by S. Kitabata (MUCC 2604); Theobroma cacao - Japan, Tokyo, Ogasawara, Chichijima Island, Oct 2011, by T. Sato (MAFF 243205 = MUCC 2746).
Note: On the resultant phylogenetic tree, five isolates examined in this study were in the same clade as L. theobromae (Fig. 1). An observation of their morphological characteristics revealed that the size and L/W ratio of matured conidia of these isolates fell into the range of L. theobromae. Lasiodiplodia theobromae has been isolated from various plants, mainly in the tropical and subtropical regions, as the pathogen of stem-end rot disease. In this study, the Japanese cultures isolated from two hosts, M. indica and T. cacao, were confirmed on the basis of a narrow sense of L. theobromae.
Lasiodiplodia sp. 1. Supplementary Fig. S4.
Conidiomata formed on PNA within 26-39 d, pycnidial, globose to oblate, thick-walled, uniloculate, light brown to dark brown, covered with hyphae; paraphyses hyaline, rounded at the apex, aseptate; conidiogenous cells hyaline to pale brown, cylindrical or rod-shaped, sporulating holoblastically, 7.5-12 × 1.8-4.5 µm, L/W = 3.09 (n = 16). Conidia ellipsoidal or rarely oblong, apex and base rounded, initially hyaline, with granular content, aseptate, becoming light brown to dark brown and one-septate after releasing from the conidiogenous cells, with longitudinal striations, 19-26 × 11-15 µm, L/W = 1.79 (n = 117, MUCC 2735).
Cultural characteristics: on PDA: colonies with aerial mycelia, light gray, or light brown, colonies reaching 90 mm diameter 3 d after inoculation, growing in the range 20-35°C. The optimum growth temperature varies in the range 25 °C-30 °C (MUCC 2735) and 20 °C-30 °C (MUCC 2739).
Isolates Examined: ; Laburnum anagyroides - Japan, Tochigi, Ashikaga, Dec 2004, by T. Kobayashi (MAFF 411002 = MUCC 2745); Anodendron affine - Japan, Okinawa, Okinawa Island, Feb 1993, by H. Nago (MAFF 306517 = MUCC 2744); Phoenix roebelenii - Japan, Tokyo, Hachijojima Island, May 1990, by J. Takeuchi (MAFF 242322 = MUCC 2731); Citrus sinensis - Japan, Tokyo, Ogasawara, Chichijima Island, 1986, by T. Sato (MAFF 306027 = MUCC 2739); Crinum asiaticum - Japan, Tokyo, Ogasawara, Chichijima Island, Sep 2013, by T. Sato (MAFF 244435 = MUCC 2736); in the flesh water, Japan, Tokyo, Ogasawara, Chichijima Island, Jun 2009, by S. Uzuhashi (MAFF241893 = MUCC 2728); Passiflora edulis - Japan, Tokyo, Ogasawara, Chichijima Island, Apr 2003, by T. Ono (MAFF 242320 = MUCC 2730); Wollastonia dentata - Japan, Tokyo, Ogasawara, Chichijima Island, Sep 2012, by T. Sato (MAFF 243711 = MUCC 2735).
Note: On the resultant phylogenetic tree, eight Japanese isolates did not form a well-supported clade with ML-BS and BI-PP (Fig. 1). Although L. thailandica, L. chiangraiensis, and Japanese isolates have similar sizes of conidia, L. chiangraiensis is characterized by conidia remaining hyaline even after maturity (Wu, Dissanayake, Chethana, Hyde, & Liu, 2021). In contrast, the Japanese isolates and L. thailandica have colored conidia after the maturity. Japanese isolates also showed differences in nucleotide sequences in each region; L. thailandica: 1-3/387 bp in ITS, 1-12/262 bp in tef1, 1/422 bp in tub2; L. chiangraiensis: 1-3/387 bp in ITS, 4/262 bp in tef1, 1/422 bp in tub2, 2/525 bp in rpb2. Additionally, all examined isolates, except MUCC 2739, had longitudinal striations on the conidia after the maturity (Supplementary Fig. S4F), which are observed in L. thailandica. In contrast, MUCC 2739 showed grid-like arrangements on their surface (Supplementary Fig. S4I), which might be a distinguishing feature. Further studies are needed to decide their taxonomical position.
Japanese isolates of L. theobromae, L. parva, and L. pseudotheobromae were reidentified as five species and one ambiguous phylogenetic species. Of them, three species, L. brasiliensis, L. hormozganensis, and L. thailandica, were newly added to the Japanese mycoflora. These results suggest that an updated taxonomical review of Japanese Lasiodiplodia species from various host plants in previous studies is needed.
The examined materials collected from the Ryukyu archipelago and Ogasawara Islands showed the rich species diversity of the genus Lasiodiplodia. The distribution and exchange of biota of volcanic or elevated coral islands, such as the Ryukyu archipelago (Kizaki, 1978; Kizaki, 1986), are significantly affected by sea-level changes and ocean currents (Flessa, 1980). Alternatively, oceanic islands, such as Ogasawara Islands, have no interaction with living things other than those drifted by ocean currents and transported by humans (Tomiyama, 1998). Although discovering a unique Lasiodiplodia species inhabiting these islands is expected, several examined isolates were the species previously reported from tropical and subtropical regions, including Southeast Asia. This may be due to the wide spreading of host plants because of human mobility. Conversely, “Lasiodiplodia sp. 1” in our study were also recognized, thereby suggesting the presence of unique species in the southern islands of Japan.
Lasiodiplodia brasiliensis is often isolated from a common host in tropical areas (Coutinho et al., 2017; Dou et al., 2017). In this study, isolates from M. indica and C. papaya were identified as L. brasiliensis and it is newly added to Japanese mycoflora. In addition, L. theobromae sensu stricto and L. thailandica were also isolated from mangoes in Miyako Island in this study. These results reveal that several Lasiodiplodia species infect Japanese mangoes even though they are cultivated on one island.
In conclusion, re-examining Japanese Lasiodiplodia isolates based on their morphological characteristics and phylogenetic analysis revealed a high degree of species diversity in Japan. However, many isolates still could potentially be misconstrued as L. theobromae sensu lato or Lasiodiplodia sp. To understand the diversity of Lasiodiplodia in Japan, further molecular phylogenetic analysis and comparative studies on their morphological characteristics are warranted.
The authors declare no conflicts of interest. All the experiments undertaken in this study comply with the current laws of the country where they were conducted.
We thank Prof. Keiichi Motohashi, Faculty of International Agricultural Development, Tokyo University of Agriculture (TUA), and Prof. Hidehiko Kikuno, Miyako Subtropical Farm, TUA, for guiding the collection of specimens in Miyako Island. This study received financial support from a JSPS KAKENHI grant (no.17K07837, no.20J12025, and no.20K06146).
