Neocosmospora caricae sp. nov. and N. metavorans, two new stem and trunk canker pathogens on Ficus carica in Iran

During 2018–2021, a survey was conducted in rainfed fig (Ficus carica L.) orchards throughout the Fars Province of Iran to investigate the occurrence of canker diseases, and to identify the causal organisms. Morphological and cultural characteristics, as well as multilocus phylogenetic analyses of the internal transcribed spacer (ITS) region of rDNA, RNA polymerase II second largest subunit (RPB2), and the translation elongation factor 1-alpha (TEF1), revealed that the recovered isolates from the infected fig trees clustered in clade 3 of Neocosmospora (Nectriaceae), including N. metavorans, and a new taxon described here as N. caricae sp. nov. Neocosmospora caricae is characterised by falcate, multiseptate, gently dorsoventrally curved macroconidia with poorly developed foot-shaped basal cells, ovoid, aseptate microconidia that cluster in false heads, and abundant terminal or intercalary chlamydospores. Pathogenicity tests indicated that isolates of both Neocosmospora species were pathogenic, causing stem canker and wood discolouration on fig saplings of “Sabz” and “Shah Anjeer” cultivars. The present study adds to existing knowledge on the aetiology of fig stem and trunk canker, and may provide essential information for developing effective integrated management strategies against canker diseases affecting fig orchards in Iran.


Introduction
The common fig (Ficus carica L.) is an ancient crop species belonging to the Moraceae family originating from the Mediterranean basin (Berg 2003). Iran is the fifth largest producer of figs after Turkey, Morocco, Greece, and Spain (FAOSTAT 2020) (Bolboli et al. 2022).
During a recent survey to identify fungal pathogens associated with canker diseases of edible fig trees in southern Iran, several Neocosmospora spp. isolates (formerly Fusarium solani species complex = FSSC) were obtained from infected tissues. Neocosmospora is one of the fusarioid genera that has been segregated from the genus Fusarium sensu lato (Lombard et al. 2015). Species of this genus affect an extensive range of hosts, including humans, animals, and plants (O'Donnell et al. 2008;Lombard et al. 2015). Several species of Neocosmospora cause stem and trunk canker diseases of trees.   (Estahban, Firuzabad, Jahrom, Kazerun, and Nayriz Counties). Transverse sections of infected branches and trunks were prepared, and small pieces (5 × 5 mm) from the margins between healthy and discoloured or decayed wood tissues were cut, washed under running tap water, surface disinfected for 1 min in a 70% ethanol, 1 min in a 2% sodium hypochlorite solution and rinsed twice in sterile distilled water (Gonzalez-Dominguez et al. 2016). Surface disinfected tissue samples were dried in sterile paper towels under a laminar flow-hood, and subsequently plated on Petri dishes containing potato dextrose agar (PDA; extract of 300-g/L boiled potato, 20-g/L glucose monohydrate, 15-g/L agarose, and distilled water) amended with tetracycline (1 mg/L). Plates were incubated at 25°C for 7 days. All isolates were then transferred onto water agar (WA; 20-g/L agar, and distilled water) and single conidial isolates established once sporulating.

Morphological characterisation
Isolates were transferred onto carnation leaf agar (CLA) (Fisher et al. 1982), oatmeal agar (OA; extract of 30-g/L boiled oatmeal, 15-g/L agar, distilled water), and PDA. Morphological identification and characterisation for all fusarioid isolates were performed based on Crous et al. (2021). Average growth rates at 25 and 30°C were obtained from colony diameters on PDA (90 mm Petri dishes with 25 ml medium), after 7 days of incubation in the dark with three replicates per isolate. Colony morphology and pigments were recorded after 7 days of incubation at 25°C in the dark (Sandoval-Denis et al. 2019), using the colour chart of McKnight and Rayner (1972).

DNA extraction, PCR amplification, and sequencing
Total fungal DNA was extracted using the method described by Mirsoleimani and Mostowfizadeh-Ghalamfarsa (2013). Mycelia were harvested from the isolates grown in potato extract broth (extract of 300-g/L boiled potatoes in distilled water) for 7-10 days, then freeze-dried, and DNA was extracted with DNG-PLUS extraction kit (CinnaGen, Tehran, Iran). DNA quality was examined with a MD-1000 Nanodrop spectrophotometer (NanoDrop Technologies, Delaware, USA). The nc rDNA internal transcribed spacer (ITS) region (ITS1-5.8S-ITS2) was amplified using the primer set ITS1 (5′-TCCTCCGCTTATTGATATGC-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC -3′) following the protocol of White et al. (1990). RNA polymerase II second largest subunit (RPB2) was amplified using primers RPB2-5F2 (5′-GGGGWGAYCAGAAGAAGGC-3′) (Sung et al. 2007) and fRPB2-7cR (5′-CCCATRGCTTGTYYRCCCAT-3′) (Liu et al. 1999) and translation elongation factor 1-alpha (TEF1) w a s a m p l i f i e d w i t h p r i m e r s E F -1 H ( 5 ′ -A T G G G T A A G G A R G A C A A G A C -3 ′ ) and EF-2T (5′ -GGARGTACCAGTSATCATG-3′) (O'Donnell 1998). Temperature and time conditions for PCR amplification are listed in Table 1. PCR amplifications were performed on a Peltier Thermal Cycler (Techne, Germany). PCR products were sequenced with the same primer pairs used for amplification by a dye terminator cycle (Cardiogenetic Research Center, Tehran, Iran). Sequenced data were deposited in GenBank (www.ncbi.nlm.nih.gov/genbank). Accession numbers are listed in Table 2.
To reconstruct the phylogenetic trees, Bayesian inference analyses on individual and concatenated ITS, RPB2, and TEF1 loci were carried out with MrBayes v. 3.1 (Ronquist and Huelsenbeck 2003). Additional sequences included in this study were retrieved from GenBank and sequences of the ascomycete Geejayessia atrofusca (Schwein.) Schroers & Gräfenhan (NRRL 22316) served as the outgroup taxon in all analyses included (Supplementary Table 1 (Nylander 2004). Two independent runs of Markov chain Monte Carlo (MCMC) using four chains were run over 1,000,000 generations. Trees were saved each 1000 generations, resulting in 10,001 trees. Burn-in was set at 25% generations. In order to conduct a phylogenetic comparison, maximum likelihood estimation was carried out using PHYLIP DNAML (Felsenstein 1993) with the same dataset. The robustness of the maximum likelihood trees was estimated by 1000 bootstraps. Phylogenetic trees were edited and displayed with TreeGraph (Stöver and Müller 2010).

Pathogenicity tests
Pathogenicity tests were conducted on detached woody shoots (fresh vegetative shoots, collected from 5-10-yearold fig trees and cut into 25-30 cm pieces (5-9 mm diam)) and mature 1-year-old fig saplings of Ficus carica cv. Shah Anjeer and cv. Sabz grown from cuttings in greenhouse conditions at 26 ± 3°C. For both experiments, the outer bark at the inoculation site was cleaned and surfacesterilised with 70% ethanol, and a 6-mm wound was made using a sterilised cork-borer. A 6-mm diam mycelium plug taken from the margin of a 5-day-old PDA culture was inserted into the wound and covered with Parafilm (USA, Bemis Packaging) to prevent desiccation and contamination. Non-colonised PDA agar plugs served as the negative control (Roux et al. 2007). In the detached woody shoots experiment, the bases of inoculated shoots were inserted into Erlenmeyer's flasks covered with Parafilm, with 500 ml of sterilised water, then kept under greenhouse conditions at 25 ± 2°C. Inoculated detached shoots and saplings, as well as uninoculated controls, were returned to the laboratory 21 days after inoculation, their bark removed, and disease symptoms investigated. For reisolation of fungal pathogens, five pieces (2 × 5 mm) from the margins of necrotic lesions were surface disinfected for 1 min in 70% ethanol, followed by 1 min in a 2% sodium hypochlorite solution, rinsed twice in sterile distilled water, and plated on PDA plates to recover and identify the inoculated fungi and complete Koch's postulates.

Field surveys and disease symptoms
Fig trees attacked by canker-causing fusarioid fungi displayed external and internal symptoms. External symptoms included leaf yellowing and defoliation, limb dieback, and three types of trunk cankers (Figs. 1 and 2). Type B cankers originated from the crown and developed upward ( Fig. 2A), whereas type C was observed as well-developed sunken trunk lesions (Fig. 2E) and type D which consisted in cracked, discoloured, and dead areas on the main stem and branches (Fig. 1B). Internal symptoms included brown to dark brown discolouration of vascular tissues and different types of wood necrosis ( Figs. 1 and 2). The occurrence of each symptom varied in an orchard from tree to tree, depending on cultivars,   , both of which were clustered strongly (1/100%) in a monophyletic subclade within N. metavorans (Fig. 3).
Several isolates with unique morphological features were recovered from trunks and branches of infected fig trees in plantations of southern Iran. BLASTn searches in GenBank showed that RPB2 sequences of these isolates had ca. 99% identity with isolates previously described as N. parceramosa  (Walther et al. 2017)). The TEF1 sequences of these isolates also had 98% identity with isolates previously identified as Fusarium sp. (strain NRRL 13414 GenBank accession No. MK818415 (Carrillo et al. 2020)) and N. petroliphila (strain NRRL 44904, GenBank accession No. KJ867424 (Ersal et al. 2015)). Furthermore, the partition homogeneity test between ITS, RPB2, and TEF1 loci resulted in a P value of ca 0.9 indicating statistical congruence, so the null hypothesis of congruence is accepted (P≥0.05), which means these genes have co-evolved.
Colony characteristics: Colonies on PDA growing in the dark with an average radial growth rate of 6.1-6.3 mm/days at 25°C, reaching 64.3 mm diam in 7 days at 25°C; white, pale luteous to luteous at centre, flat to slightly raised, cottony, with abundant aerial mycelium; colony margin filiform. Reverse pale straw to pale luteous. On OA incubated in the dark reaching 61.2 mm diam in 7 days at 25°C; white to yellowish, flat, membranous with scant white aerial mycelia.
Other specimens examined (

Pathogenicity tests
Pathogenicity of representative isolates Esh191B, ES212, and ES216 were evaluated in two experiments on detached twigs and 1-year-old saplings, respectively. All isolates used in both pathogenicity tests produced cankers, vascular tissue discolouration and yellowing on Ficus carica cv. Shah Anjeer and cv. Sabz saplings. The first visible symptom was the appearance of discolouration that began from the inoculation site and developed longitudinally on detached twigs and saplings. Based on pathogenicity tests, N. metavorans and N. caricae sp. nov. isolates produced canker disease symptoms on fig stems 10 and 21 days after inoculation, respectively (Fig. 5). Common symptoms included brown to dark brown discolouration of vascular tissues, wood necrosis, and branch dieback. Yellowing and defoliation of sapling were observed 5 months after inoculation. Symptoms were similar to those observed in infected fig trees in orchards. Inoculated isolates could be recovered from lesion margins. Control plants remained healthy.

Discussion
The  (Droby et al. 2011, Kosoglu et al. 2011Guarnaccia et al. 2021), and F. proliferatum (Fawzi 2003). It seems that F. proliferatum isolates from many crops, including fig trees, are phylogenetically different from the original ex-type strain, and belong to a morphologically and phylogenetically diverse clade, F. annulatum Bugnic (Yilmaz et al. 2021).
Multi-locus phylogenetic analyses using three loci (ITS, RPB2, and TEF1), as well as morphological analysis, revealed that all fusarioid isolates in this study belong to clade 3 of the genus Neocosmospora, including N. metavorans and a new taxon, N. caricae sp. nov. Sandoval-Denis et al. (2019) provided a comprehensive phylogeny for N. metavorans, which included 19 isolates that originate from different substrates, namely humans, insects, and plants. These isolates are clustered in several subgroups in the clade. They are mostly known from human clinical samples, and only a single isolate Isolates of N. metavorans were also recovered from the intestines and mouth parts of Phryneta spinator larvae. This longhorn beetle from Cerambycidae is a wood borer that attacks fig trees in Iran. The larvae tunnels were also  (Freeman et al. 2013), and N. metavorans isolates from the guts of the wood-boring cerambycid beetles, Anoplophora glabripennis Motschulsky (Herr et al. 2016). Hence, fig tree borer larvae can be considered as potential vectors or transmitters of canker-causing Neocosmospora species in fig. More experiments, however, should be conducted to confirm this hypothesis.
Several N. caricae sp. nov. isolates were recovered from trunks and branches of infected fig trees in plantations of southern Iran. Morphological and multigene phylogenetic studies using ribosomal and protein-coding loci (ITS, RPB2, and TEF1) showed that these isolates were significantly distinct from other known Neocosmospora species. The differences were more evident in the TEF1 phylogeny than in the other genes. Neocosmospora caricae sp. nov. appeared as a sister taxon to N. petroliphila, one of the most prevalent species associated with human infections (Sandoval-Denis et al. 2019). Morphologically, the apical cells of sporodochial conidia in N. caricae sp. nov. were short, and the basal cells poorly developed foot-shaped, vs longer and more curved apical cells of sporodochial conidia in N. petroliphila. Furthermore, sporodochial conidia in N. caricae sp. nov. were shorter than those of N. petroliphila and N. metavorans (Short et al. 2013). The morphological differences, as well as the phylogenetic analyses, supported describing these isolates as a new species.
Four different types of canker were observed in the infected fig orchards; we named them as types A-D (Bolboli et al. 2022). Only the previously reported Diaporthe cinerascens (syn. Phomopsis cinerascens) (Banihashemi and Javadi 2009) was recovered from the type A cankers: trunk lesions with zonation. Our observations, combined with these results, revealed that the fig canker-causing Neocosmospora isolates can induce types B, C, and D cankers. Type B cankers that originate from the crown were more widespread than type C, with well-developed sunken lesions on the trunks, and type D, cracked, discoloured, and dead areas on the main stem and branches. However, N. caricae sp. nov. may cause type B, or C in the orchards, whereas type C and D can result from N. metavorans infections of the fig trees. Types C and D cankers were also caused by the recently described S. banihashemiana (Bolboli et al. 2022). Two types of discolouration were also observed in the transverse sections of the infected fig trees. Neocosmospora caricae sp. nov. isolates caused irregular-shaped and wedge-shaped necrosis, whereas N. metavorans necrosis was crescent-shaped and wedgeshaped in the transverse sections of infected trees.
Since Neocosmospora species could have a nonpathogenic endophytic or pathogenic lifestyle (Sandoval-Denis et al. 2019), our pathogenicity results demonstrate that both N. metavorans and N. caricae sp. nov. were pathogenic and responsible for fig stem and trunk canker. Based on our observations, these newly reported pathogens may represent a severe threat to fig plantations.
In conclusion, this study identified two new pathogenic fungal species from the Nectriaceae, N. metavorans and N. caricae sp. nov., associated with trunk and branch can-