The common fig (Ficus carica L.), a traditional fruit tree of the Moraceae family, is cultivated worldwide as a dry and fresh food (Solomon et al. 2006). In Japan, figs are mainly cultivated as fresh food and are one of the most economically important fruit trees in the Fukuoka Prefecture in northern Kyushu. The main fig cultivars in the country are ‘Houraishi’ and ‘Masui Dauphine,’ while the high-quality fig cultivar ‘Toyomitsuhime’ was developed by the Fukuoka Agriculture and Forestry Research Center in 2006 and is now commonly cultivated in the Fukuoka Prefecture.
In August 2007, black and withered leaves on ‘Toyomitsuhime’ fig trees and severe leaf fall were observed in several commercial orchards in Fukuoka Prefecture (Fig. 1a). The symptoms were similar to those of black leaf blight in fig (Inoue et al. 2008). In addition, fruit rot, twig blight, branch canker, and dieback, which were not reported by Inoue et al. (2008), were observed in fig trees in the same orchards. The fungus causing black leaf blight was morphologically identified as Fusicoccum aesculi (Inoue et al. 2008) and then as Neofusicoccum parvum (Kikuhara and Inoue 2011). The identification of the genus and species in Botryosphaeriaceae via morphological analysis alone was insufficient and required molecular genetic analysis (Crous et al. 2006). This study aimed to add new symptoms to black leaf blight in fig and reidentify the pathogen using morphological and multilocus phylogenetic analysis.
From 2007 to 2009, the symptoms of fig trees (‘Toyomitsuhime’) were surveyed in six orchards, including the orchard in Fukuoka Prefecture mentioned above. From spring to summer, orange-brown spots initially appeared on petioles and veins of leaves, some of which became black-brown fusiform lesions (Fig. 1b, c) and spread throughout (Fig. 1d, e), after which the leaves withered and fell off (Fig. 1a). When the lesion reached the node (Fig. 1f), a dark brown lesion formed on the twig (Fig. 1g). The lesion became a fusiform canker on the branch in the following year (Fig. 1h). Unlike the change in symptoms described above, lesions spread from the cut end-pruned branches (Fig. 1i). In young fruits, orange-brown spots appeared in the summer (Fig. 1j). Some spots became red-brown spots—approximately 5 mm in size—at the harvest stage. From late summer to autumn, the fruits infected at the pre-harvest stage rotted, turning from dark yellow to black (Fig. 1k), and then dried and shrunk (Fig. 1l).
Several diseased leaves, branches, and fruits were collected from the orchards mentioned above for isolation. Small sections of petiole lesions were sterilized with 1% sodium hypochlorite for 1 min and 70% ethanol for 30 s, rinsed twice with sterile distilled water, air-dried, and placed on potato dextrose agar (PDA) (Difco Laboratories, Detroit, MI, USA). In addition, internal sections of diseased branches and fruits were removed using a flame-sterilized knife and placed on PDA. Furthermore, the inside of the ascomata on the diseased branches was scooped out while being observed under a stereomicroscope and spread on water agar. The ascospore was taken out and grown on PDA at 25℃. The same type of colony is frequently isolated from several symptomatic leaves, branches, fruit tissues, and ascospores. Representative isolates from their sources, namely, FFNP0901 (MAFF 243818, NBRC 115620), FFNP0904 (MAFF 243819, NBRC 115621), FFNP1002 (MAFF 243820, NBRC 115622), and FFBP1102 (MAFF 243821, NBRC 115623), were used in this study.
Three types of pathogenicity tests were performed using 5- to 7-year-old fig trees (‘Toyomitsuhime’) planted in pots 60 cm in diameter in a greenhouse. Inocula of all tests used mycelial plugs of three or four isolates grown on PDA for 7-10 d at 25℃. PDA plugs were used as negative controls. First, inocula of FFNP0901, FFNP0904, FFNP1002, and FFBP1102 were placed on the petioles of six young leaves and covered with Parafilm M for 2-3 d. Typical dark brown lesions were observed on all inoculated petioles 7 d after inoculation (Fig. 1n). Furthermore, some lesions on the petiole spread to twigs 40 d after inoculation, forming dark brown irregular rhombic lesions at the nodes (Fig. 1o, p). The lesions resembled the symptoms of the pre-growth branch cankers observed in the field. Second, each inoculum of FFNP0901, FFNP0904, and FFNP1002 was placed on more than six young fruits 3-4 cm wide and wrapped with Parafilm M for 2-3 d. In the inoculation of young fruits, brown spots were formed 1 week after inoculation, some of which spread, and fruits fell or rotted. The fruits inoculated at the pre-harvest stage rotted and turned black 3-4 d after inoculation (Fig. 1q). The inoculated fruits showed symptoms similar to those observed in the field. Then, each inoculum of FFNP0901, FFNP0904, and FFNP1002 was placed on six green shoots wounded into a 7-mm square using a cutter knife and wrapped with Parafilm M for 2-3 d. Dark-brown lesions were observed in all inoculated shoots 7 d after inoculation (Fig. 1q). The fungi reisolated from the representative lesions of each inoculation were similar to those of the inoculated isolates. The lesions formed by the inoculation of the twigs and the symptoms of dieback spread. Thus, Koch’s postulates were fulfilled. Inoue et al. (2008) reported that the only symptom caused by the fungus was leaf blight. This study is the first to report the symptoms fruit rot, twig blight, branch canker, and dieback caused by the causal agent of black leaf blight in fig.
Morphological characteristics of the fungus were investigated. Pycnidia and ascomata were observed on the surface of the branch lesions under a stereomicroscope. The conidia, asci, and ascospores were observed and measured using an optical microscope. In addition, the isolates were cultured on sterilized fig twigs in blacklight at 25℃ for 2 weeks, and the pycnidia and conidia that formed were measured. To examine cultural characteristics, the isolates were placed on PDA and OA and observed in the dark at 25°C for 14 d. The growth rate of mycelia at each temperature was measured. Mycelial discs of each isolate cut from the edges of the colonies on PDA were placed on a new PDA plate, cultured at 5–40°C in the dark at 5°C increments, and incubated for 2–7 d. The test was performed twice.
Partial sequences of the internal transcribed spacers (ITS) of the ribosomal RNA, translation elongation factor 1-α (EF1-α), beta-tubulin (TUB2), and RNA polymerase II (RPB2) genes in these fungi and fig2-1 isolated in Okayama (Inoue et al. 2008) were analyzed. First, total genomic DNA was extracted from fungal mycelia grown on PDA using the MagExtractor -Plant Genome- kit (Toyobo, Osaka, Japan). Polymerase chain reaction (PCR) was carried out using the primers ITS1/ITS4 (White et al. 1990), EF1-688F/EF1-1251R (Alves et al. 2008), Bt-2a/Bt-2b (Glass and Donaldson 1995), and RPB2-5f/RPB2-7R (Liu et al. 1999). PCR was performed using AmpliTaq Gold 360 Master Mix (Applied Biosystems, Foster, USA) with the following conditions: 95°C for 2 min; 30 cycles at 95°C for 1 min, 55°C for 1 min, and 72°C for 1 min; and 72°C for 5 min. PCR products were confirmed via 2.0% (w/v) agarose gel electrophoresis in Tris-borate-EDTA buffer and purified using the GenElute PCR Clean-Up Kit (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. Direct sequencing of the purified products was performed by Sigma-Aldrich Japan Corporation (Tokyo, Japan) or Eurofins Genomics Inc. (Tokyo, Japan) using forward and reverse primers. Sequences lengths of approximately 550-bp, 250-bp, 300-bp, and 850-bp were determined for the ITS, EF1-a, TUB2, and RPB2 PCR amplicons of five isolates, respectively. The closest relatives to the sequences of isolates in this study were searched against the NCBI GenBank database using the Basic Local Alignment Search Tool. Consequently, the ITS, EF1-α, TUB2, and RPB2 sequences of the isolates in this study were similar in identity to Neofusicoccum species, although they did not match specific species. Ex-type isolates or isolates with available sequences for the four loci were selected as representative isolates from each Neofusicoccum species for phylogenetic analysis. Sequences of the four loci of 47 strains and ITS sequences of 62 strains were obtained from NCBI for phylogenetic analysis (Supplementary Table 1). Sequences from the isolates in this study were aligned with those belonging to the Neofusicoccum genus and the outgroup Botryosphaeria dothidea isolate CBS 100564 using MEGA X. The sequence (1749 bp) was created by combining and aligning ITS (540 bp), TUB2 (330 bp), EF1-α (292 bp), and RPB2 (587 bp). A unique position in the sequence of this fungus was discovered. The positions 72 and 164 of TUB2 were A and T in this fungus, respectively, but were G and C in all other species of Neofusicoccum. Phylogenetic analysis was performed with each ITS and the connection of the four loci (ITS, EF1-α, TUB2, and RPB2) using maximum likelihood (ML) and maximum parsimony (MP) in MEGA X (Kumar et al. 2018). The best evolutionary model for ML was determined using MEGA X based on minimum Bayesian Information Criterion scores. ML trees of ITS sequences and the concatenated sequences of the four loci were constructed with the Kimura 2-parameter model (gamma distributed) selected as the best evolutionary model for ML. The consistency and retention indices were calculated using MEGA X in the MP. MP trees of ITS sequences and the concatenated sequences of the four loci (length = 167 and 532, respectively) were constructed, with consistency indices of 0.432203 and 0.652256, respectively and retention indices of 0.804646 and 0.801775, respectively. The support values for ML and MP branches were determined using 500 bootstrap replicates. The ML and MP phylogenetic trees based on the ITS sequences were topologically similar (Fig. 3). Some ML and MP bootstrap values were high and strongly supported by the nodes. Our isolates were separated into a monophyletic clade different from the N. parvum-N. ribis species complex and the others by phylogenetic trees generated using either method. In addition, a similar phylogenetic analysis was performed using a combination of sequences of the four loci. The ML and MP phylogenetic trees were nearly identical in topology (Fig. 4). The ML and MP bootstrap values of the phylogenetic trees based on the four loci were higher than those of the phylogenetic trees based on the ITS region. The clade including our isolates was independent of N. parvum-N. ribis species complex and others.
The fungus that causes fig black leaf blight was unrelated to the known Neofusicoccum species based on the phylogenetic analysis of the concatenated sequences of the four loci (ITS, EF1-α, TUB2, and RPB2), even though it was morphologically similar to N. parvum. Moreover, it does not belong to the N. parvum-N. ribis species complex. The phylogenetic relationship of Botryosphaeriaceae has been shown by molecular analysis, and the morphological description is supplementary (Phillips et al. 2013). We determined that this fungus is a new species.
Several diseases of fig are known to be caused by fungi belonging to the Botryosphaeriaceae family. In Japan, Macrophoma sp. causing fruit rot and Lasiodiplodia theobromae causing leaf fall have been reported (Abe and You 1959; Saitou et al. 2018). However, a disease caused by Neofusicoccum species has not been reported. Canker and twig blight caused by N. parvum on F. carica has been reported in Italy (Aiello et al. 2020). The pathogenicity of N. parvum in this study differed from that reported by Aiello et al. (2020), owing to the differences in the cultivars of each test plant. Although the fungus identified as N. parvum was isolated from parts with the symptom of black leaf blight in Okayama Prefecture coincidentally, it had little pathogenicity to fig (Inoue et al. 2008). In addition, fungi isolated from leaves, branches, and fruits have never been identified as N. parvum in Fukuoka Prefecture. The fungi used in this study differed from N. parvum in terms of pathogenicity. Therefore, we propose that the fungus causing black leaf blight disease in fig is a new species and causes fruit rot, twig blight, branch canker, and dieback.
Taxonomy
Neofusicoccum ficicola Kikuhara, Motohashi, and Inoue sp. nov. Figs. 1, 2
Mycobank
Etymology: Named for fig (Ficus), which the fungus inhabits (-cola).
Typification: Japan. Fukuoka, a fig orchard on symptomatic leaves of F. carica “Toyoumitsuhime,” 1999, K. Kikuhara, FFNP9901 (holotype, TFM FPH-10463, ex-type culture FFNP9901 = MAFF 243818 = NBRC 115620).
Sexual morphology: The perithecium was on the surface of branch cankers born last year. It was globose with short conical papilla, smooth, black with white content, and 141–214 μm in size (Fig. 2a). Asci were clavate and 71–133×16–20 μm in size (Fig. 2b). Ascospores were broadly ellipsoidal to fusoid, smooth, hyaline, and 15–20×7–11 μm in size (ave. 18.3×8.8 μm) (Fig. 2c). Asexual morphology: The pycnidia were on the surface of petiole lesions, branch cankers, and rotten fruit (Fig. 2a). They were black, globose, non-papillate, and 64–200 μm in size (Fig. 2e). Conidia were hyaline, fusiform to ellipsoid, and 15–19×5–8 μm in size (ave. 17.3×6.4 μm). The artificially formed pycnidia were black, globose, and non-papillate and could not be measured by a crowd (Fig. 2h). The artificially formed conidia were hyaline, fusiform to ellipsoid, 12–20×5–7 μm in size (ave. 16.0×6.0 μm), and turned light brown with 1–2 septa with age (Fig. 2f, g).
Cultural characteristics: On PDA and OA at 25 °C in the dark, the colonies were initially white-gray, turned gray after 7 d (Fig. 2i, j), and turned dark gray after 14 d. The fungus rapidly grew at 25 to 30°C and barely grew at temperatures of 10°C or below and 35°C or more (Fig. 2). The highest mycelial growth rate was 9–12 mm/d at 25°C.
Habitat: On leaves, fruits, twigs, branches, and trunks of F. carica, causing blight and canker.
Known distribution: Western Japan.
Note: Shape and size of perithecium, asci, ascosore, pycnidia, and conidia in this species is similar to those of N. parvum. The species cannot morphologically differentiate from N. parvum-N. ribis species complex. The fungus forms pycnidia on the leaves, fruits, and twigs of F. carica. It forms perithecia on branches that were affected last year. Other species of the genus Neofusicoccum are not known to readily form pycnidia No perithecium of them has been found. However, it is distinguished from other species of Botryosphaeriaceae by phylogenetic tree analysis of rDNA-ITS, EF1-α, TUB2, or RPB2 sequences. In addition, positions 72 and 164 of TUB2 were A and T in this fungus, respectively, but were G and C in all other species of Neofusicoccum.