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Cryphonectria carpinicola discovered in Japan: first report of the sexual state on Carpinus tree
2023 Volume 64 Issue 5 Pages 123-127
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2023 Volume 64 Issue 5 Pages 123-127
Cryphonectria carpinicola is an ascomycetous fungus that has been regularly found in its asexual form on European hornbeam (Carpinus betulus) in Europe over the past two decades. Here we describe the discovery of C. carpinicola in Japan and report for the first time its sexual state on Carpinus species. No symptomatic trees were observed, but stromata were found saprotrophically on broken branches of Carpinus species on the forest floor. The sexual structures of C. carpinicola resembled that of other Cryphonectria species and strongly resembled those of the closely related species C. radicalis. A phylogenetic tree based on the internal transcribed spacer sequences showed monophyly for the Japanese and European isolates of C. carpinicola. Further studies on the distribution and host range of C. carpinicola in Japan and on the life history strategies of this fungus are needed.
Scientists around the world have historically deposited biological material in curated herbaria, fungaria or culture collections, preserving an invaluable resource for future scientific study. Such preserved biological material includes the first documented living strain JS13 of a fungus that would not receive its species name until twenty-three years after its collection in 1998 (Liu, Linder-Basso, Hillman, Kaneko, & Milgroom, 2003).
The long history of naming began at the same time strain JS13 was collected, but on the other side of the globe, when Italian tree pathologists described declining hornbeams in the late nineties, reportedly infected by an unidentified Endothiella sp., obsolete name for anamorphs of the genus Cryphonectria according to the Melbourne Code (McNeill et al., 2012). From then on, reports of the decline of the European hornbeam (Carpinus betulus) followed continuously, first from various northern Italian cities, later from Austria, Germany, Switzerland, and from the easternmost limit of the European hornbeam's distribution in Iran (Cornejo, Hauser, Beenken, Cech, & Rigling, 2021). In addition to Cryphonectria sp., another fungus identified as Anthostoma decipiens was found to be involved in the decline phenomenon (Rocchi, Quaroni, Sardi, & Saracchi, 2010). The long naming history of the second fungus came to an end when it was described as the novel species Cryphonectria carpinicola (Cornejo et al., 2021).
The strain JS13 was collected in 1998 from symptomatic Japanese chestnut (Castanea crenata) in the locality Jise of Kyoto Prefecture during an extensive sampling campaign to study the causal agent of the chestnut blight disease, Cryphonectria parasitica, and associated mycoviruses (Liu et al., 2003; Liu, Dynek, Hillman, & Milgroom, 2007). Bark samples with stromata and only a few samples without stromata but from symptomatic chestnut trees were collected. Isolations resulted in 472 exemplars of C. parasitica and 44 C. nitschkei, an obsolete synonym for C. japonica (Gryzenhout, Wingfield, & Wingfield, 2009). Even then, C. japonica was known to have a wide host range in Japan from Fagaceae, Pinaceae to Betulaceae (Kobayashi & Ito, 1956; Kobayashi, 1970). In addition, viral dsRNA has been detected in strain JS13 (Liu et al., 2007), which can alter the appearance of the mycelium. Therefore, it was assumed that this viral dsRNA was responsible for the peculiar colony morphology of JS13, whereas the possibility that it might be an undescribed fungal species was not considered at this time. Consequently, strain JS13 was later deposited in the American Type Culture Collection (ATCC) under the name Cryphonectria nitschkei and identification number MYA-4104 along with the notation ‘contains novel virus’. However, in later studies on this virus, the fungal barcode of JS13 indicated that this exemplar did not belong to the species C. japonica but was closely related to C. radicalis (Shahi, Eusebio-Cope, Kondo, Hillman, & Suzuki, 2019). Only after its formal description could the species affiliation of isolate JS13 be definitively clarified as C. carpinicola twenty-three years after its collection (Sato et al., 2021).
Thanks to the preserved strain JS13 our interest was aroused in whether C. carpinicola is native to Japan, and a scientific mission to Japan by the first author opened the opportunity to conduct a short field trip. The purpose of the present study was thus to verify (i) whether C. carpinicola is more common in Japan than this incidental finding in 1998, and (ii) whether it can also occur on Carpinus species according to reports from Europe (Cornejo et al., 2021; Cornejo, Risteski, Sotirovski, & Rigling, 2023; Crampton, Pérez-Sierra, & Denman, 2022).
In Japan five Carpinus species are known; Carpinus japonica, C. tschonoskii, C. laxiflora, and C. cordata are deciduous and arboreal trees, and C. turczaninovii is a deciduous shrub tree growing exclusively on limestone mountain ridges. Carpinus japonica, C. tschonoskii, and C. laxiflora are among the major components of deciduous broadleaf forests on the Island of Shikoku in southwestern Japan, and often found at sunny slopes. Therefore, three sites in the Island of Shikoku were selected for the 26-28 Oct 2022 field trip: the Ichinomata study forest (Shimanto, Kochi), Mount Ryuoh (Manno, Kagawa), and the Tengu Highland (Yusuhara, Kochi). Although Japanese chestnut is also common in Shikoku, chestnut trees were not considered for sampling because C. parasitica was assumed to be the predominant Cryphonectria species on these trees (Liu et al., 2003). Of the three sites visited, orange Cryphonectria-like stromata were not found on living trees at all, but only on fallen small branches on the ground in the Tengu Highland Forest (Fig. 1A).
The collecting site in Tengu Highland encompasses the mountain ridges on the border between Kochi and the Ehime prefecture (1,350 m a.s.l.; 33° 28’ 36’’N, 133° 0’ 28’’E). At the Yusuhara Observatory, located 11 km southwest of the collection site and at 415 m a.s.l., the annual precipitation and the mean annual temperature are 2,729 mm and 13.4 °C, respectively (Japan Meteorological Agency, https://www.jma.go.jp/jma/indexe.html, accessed on 04-12-2022). Snowy weather is usually observed from Dec to Feb in the highland. Small twigs were collected from the ground along a trail leading from the Tenguso Hotel parking lot through a forest dominated by Abies homolepis, Fagus crenata, Quercus crispula var. crispula, Carpinus japonica, C. tschonoskii, Stewartia monadelpha, Aesculus turbinata and Acer spp. The forest floor is partially covered with dwarf bamboo (Sasa nipponica), and occasionally limestone rocks are found in the forest.
When stromata were found on fallen branches, the trees adjacent to the lying branches were visually inspected, but no disease symptoms were observed. Due to advanced autumn defoliation, only for the specimen THF5 the host tree species could be determined as C. japonica. Other hornbeam specimens were identified to a genus level. Therefore, the wood histology was checked using radial, longitudinal and transversal sections through the branches. All branches were determined to belong to Carpinus following Schweingruber (1990).
Cryphonectria-like stromata from eight branches were used for fungal isolation. Small stromata pieces were laid out on potato dextrose agar (PDA) (39 g/l; BD DifcoTM) containing 0.1 mg/ml of the antibiotic streptomycin (Sigma-Aldrich®) to prevent bacterial growth. Subsequently, the isolates were grown on PDA at 25 °C and under a 14 h light, 10 h dark regime in order to document their cultural characteristics (Fig. 1B), and to stimulate the production of asexual spores used for the long-term preservation. All isolates were stored frozen in 22% glycerol at −80 °C in the Swiss Federal Research Institute WSL culture collection (Table 1).
| Voucher | Tree host | Herbarium ID of the field samplea | Collection ID of the isolateb | GenBank accession no. |
| THF1 | Carpinus sp. | MBK-0341545 | M10783 | OQ852894 |
| THF3 | Carpinus sp. | MBK-0341546 | M10782 | OQ852895 |
| THF4 | Carpinus sp. | MBK-0341547 | M10781 | OQ852896 |
| THF5 | Carpinus japonica | MBK-0341548 | M10780 | OQ852897 |
| THF6 | Carpinus sp. | MBK-0341549 | M10779 | OQ852898 |
| THF7 | Carpinus sp. | MBK-0341550 | M10778 | OQ852899 |
| THF8a | Carpinus sp. | MBK-0341551 | M10777 | OQ852900 |
| THF9 | Carpinus sp. | MBK-0341552 | M10776 | OQ852901 |
| JS13 | Castanea crenata | n/a | ATCC:MYA 4104 | OQ852902 |
a MBK = Herbarium of the Makino Botanical Garden, Kochi City, Island of Shikoku, Japan.
b M = Culture collection of the Swiss Federal Institute for Forest, Snow and Landscape Research (WSL), Birmensdorf, Switzerland. ATCC = American Type Culture Collection, Manassas, Virginia, U.S.A.
To determine the species of isolates, DNA was extracted from mycelial cultures using the semi-automated KingFisherTM 96 Flex (Thermo ScientificTM) using LGC reagents and Kingfisher 96/Flex (LGC Genomics GmbH, Berlin, Germany), according to the manufacturer's instructions. The barcode internal transcribed spacers (ITS) of the nuclear rRNA gene cluster were amplified and sequenced using the primer pair ITS5-ITS4 according to published protocols (White, Bruns, Lee, & Taylor, 1990; Cornejo et al., 2021). The resulting forward and reverse sequences were assembled using the software DNA Main Workbench v22 (CLC bio, Qiagen). The ITS sequences of several Cryphonectria species contained long stretches of single nucleotide repeats, resulting in a rapid decline in Sanger read quality after the long poly(dA) and poly(dT) stretches. In such cases, reference sequences were used for assembling the forward and reverse reads. The ITS-sequences were verified in GenBank (www.ncbi.nlm.nih.gov, accessed on 28-12-2022) using the nucleotide BLAST search.
Sequence alignments of closely related Cryphonectria species (C. radicalis and C. naterciae) were directly downloaded from GenBank and used for a phylogenetic analysis with C. parasitica as outgroup species. Poly(dA) and poly(dT) regions produced ambiguous alignments and were thus excluded from further analyses by processing all datasets using Gblocks 0.91b (Castresana, 2000; Talavera & Castresana, 2007) on the Phylogeny.fr platform (Dereeper et al., 2008). The phylogeny was reconstructed using the maximum likelihood criterion as applied in PhyML 3.0 (Guindon et al., 2010) on the ATGC platform (http://www.atgc-montpellier.fr, accessed on 28-12-2022), using the Smart Model Selection and the Akaike Information Criterion. The resulting tree was displayed in TreeGraph 2 (Stoever & Mueller, 2010), including bootstraps percentages of 1,000 pseudo-replicates. Japanese samples were found to form a monophyletic group together with samples of C. carpinicola from Europe with 84% bootstrap support, including the ITS-barcode of the species holotype (GenBank accession, MT330391).
Additionally, the morphology of the stromata was investigated by hand sectioning of air-dried material and microscopic examination in tap water. Measurements and photos were taken with a Zeiss Discovery V8 stereo lens equipped with a Zeiss Axiocam 506 digital camera and a Zeiss Axio Scope A1 microscope equipped with a Axiocam 208 color digital camera and Zen 2.3 software (Carl Zeiss Microscopy GmbH, Germany). Asexual structures were found and documented on seven branches of Carpinus species, and ascostromata were discovered and studied in detail only on specimen THF7 of Carpinus sp. (Fig. 2).
Morphology
Cryphonectria carpinicola D. Rigling, T. Cech, Cornejo & L. Beenken, Fungal Biol. 125: 354, 2021.
MycoBank no.: MB 837752.
First description of sexual state occurring on Carpinus sp. Fig. 2 A-G: Ascostromata gregarious, pulvinate, semi-immerses in bark, pale orange, 0.2-0.8 × 0.4-1.4 mm and up to 0.8 mm high. Perithecia 2-20 per stroma, valsoid, embedded at base of stroma, black, +/- globous, 300-400 µm in diam.; necks 50-60 µm wide, black, emerging at stroma surface with ostioles imbedded in orange-brown papillae of stromatal tissue, 100-120 µm in diam. and up to 70 µm high. Asci 28-37 × 6-7(-10) µm (n = 10), fusiform, with ring-like apical ascus apparatus, 8-spored. Ascospores (6-)6.5-9(-11) × (2-)2.5-3(-3.5) µm (n = 40), 7.6 × 2.8 µm on average, length/wide ratio 2.2-5.6, 2.8 on average, colorless, hyalin, ellipsoidal to fusoid, ends round, with one median or sub-median septum, slightly constricted at septum, cell content hyalin, with small droplets. Remains of conidiomata in the same or separate stromata.
Substrate: On a dead twig, 1 cm in diam., of Carpinus sp. laying on the forest ground.
Specimen examined: JAPAN, Island of Shikoku, Tengu Highland, along the hiking trail starting from the Tengosou hotel, 33° 28’ 38.40” N, 133° 0’ 21.92” E, 1,350 m a.s.l. , 28 Oct 2022, C. Cornejo and T. Otani (THF7), ZT Myc 66431, MBK-0341550.
Conidiomata (Fig. 2 H-K) occurring on Carpinus spp. as described in Cornejo et al. (2021) but the stromata are smaller (0.2-0.9 × 0.5-1.5 mm) and with paler orange surface.
Substrate: On a dead twig, 0.5-2 cm diameter, of Carpinus japonica and Carpinus sp. laying on the ground.
Additional specimens examined containing only conidiomata: JAPAN, Island of Shikoku, Tengu Highland, along the hiking trail starting from the Tengosou hotel, 33° 28’ 38.40” N, 133° 0’ 21.92” E, 1,350 m a.s.l., Carpinus japonica (THF5), Carpinus sp. (THF1, THF3, THF4, THF6, THF8a, THF9), 28 Oct 2022, C. Cornejo and T. Otani.
Note: The sexual state of C. carpinicola described here for the first time strongly resembles those of other Cryphonectria species (Gryzenhout et al., 2009), with the closely related C. radicalis being the most similar. Its ascospores, which are (5.5-)6-7.5(-8.5) long and 2.5-3.5 µm wide (Gryzenhout et al., 2009), are only slightly shorter than those of C. carpinicola, while the ascospores of the other Cryphonectria species are clearly longer and broader (Gryzenhout et al., 2009). The stromata of C. carpinicola found on Carpinus betulus in Europe were with a size of 1-5(-10) × 0.5-1.5 mm (Cornejo et al., 2021) considerably larger than those from Japan. This difference could be related to the substrate. In Europe, C. carpinicola was found mainly on dead tree trunks and thicker branches, while the presented finds from Japan were from thinner twigs.
So far, this fungus is known only on Carpinus betulus in Europe and in the asexual conidial stage (Cornejo et al., 2021; field observations of the last author based on more than 40 collections from Switzerland, distribution atlas https://swissfungi.wsl.ch/en/index.html, accessed on 24-01-2023). The genus Cryphonectria appears to have a host preference for members of the Fagaceae, particularly the genera Castanea and Quercus (Gryzenhout et al., 2009). Despite the extensive sampling of chestnut trees in order to study the chestnut blight, C. carpinicola was never found on European chestnut, hence this fungus was considered an exception to this rule. However, our (Cornejo et al., 2021) and former inoculation tests by Saracchi, Sardi, Kunova, and Cortesi (2015), as well as voucher JS13 of C. carpinicola from Castanea crenata, confirm that this species has an affinity for Fagaceae, although it acts more as a weak parasite, as no bark cankers were observed.
For the present study, it was not possible to conduct a more intensive field excursion, but it is remarkable that the occurrence of C. carpinicola in Japan could be confirmed in this short time. Based on this positive result, we must assume that C. carpinicola is more abundant in Japanese deciduous forests than expected. Furthermore, the occurrence of the sexual state in Japan indicates a possible area of origin for this species. Considering that this fungus has been discovered in Central Europe only in the last twenty-five years, the question arises whether C. carpinicola has been introduced into Europe only recently or has been present here for a long time. Therefore, our laboratory is working on the development of highly variable C. carpinicola specific markers (Simple Sequence Repeats, SSR) that will allow detailed studies of regional genetic diversity and gene flow at different spatial scales. Future studies involving SSR markers and extensive sampling in deciduous forests will elucidate the genetic diversity of C. carpinicola and its host tree species in Japan.
The authors declare no conflicts of interest.
We are very grateful to Ayako Maeda (the Kochi Prefectural Makino Botanical Garden) for the information of suitable Carpinus sites in Kochi and Daniel Rigling for his generous support at all stages of this study. This work was supported by the Ohara Foundation for Agriculture Research.
