Stilbocrea banihashemiana sp. nov. a New Fungal Pathogen Causing Stem Cankers and Twig Dieback of Fruit Trees

Stem cankers and twig dieback were the most serious disease of fig (Ficus carica) and loquat (Eriobotrya japonica) noticed in a survey of fruit tree orchards in the Fars Province, Iran. Isolates of Bionectriaceae were consistently recovered from symptomatic fig and loquat trees. Phylogenetic analyses of multiple nuclear loci, internal transcribed spacer regions (ITS) of rDNA, RNA polymerase II subunit 2 (rpb2), and translation elongation factor 1-α (tef1), combined with morphological observations, revealed that isolates could be referred to a still unknown taxon, which was formally described as Stilbocrea banihashemiana sp. nov. Phylogenetically, isolates from fig and loquat trees clustered in a well-supported monophyletic group within the Stilbocrea clade of Bionectriaceae, closely related to S. walteri. Stilbocrea banihashemiana sp. nov. was characterized by the lack of stilbella-like asexual structure in both natural substrates and pure cultures and produced two morphologically distinct types of conidia, globose and cylindrical, formed on short and long simple phialides. In pathogenicity tests, S. banihashemiana sp. nov. induced stem cankers in both fig and loquat, wood discoloration in fig and twig dieback in loquat. Pathogenicity tests also showed that the potential host range of this novel pathogen includes other economically relevant horticultural trees.


Introduction
The edible or common fig (Ficus carica L., family Moraceae) is a deciduous species native to southwest Asia and the Mediterranean region and the loquat or Japanese medlar [Eriobotrya japonica (Thunb.) Lindl., family Rosaceae] is an evergreen species supposed to be native to China and has also grown since ancient times in southern Japan; they are adapted to subtropical to temperate climates and are widely grown worldwide. Both are fruit trees of economic relevance in Iran. The Smyrna-type fig, including the two major groups of dried and fresh figs, is one of the most important horticultural crops in Iran, with 51,000 ha in arid and semi-arid regions of the country devoted to the cultivation of this fruit tree [1,2]. Iran is the fourth largest fig producer country in the world, after Turkey, Morocco, and Greece, and produces annually 56,557 tons of figs [3]. About 90% of fig-cultivations area in Iran is located in Fars Province. This region is also the third loquat producer in Iran, with an annual production capacity of 432 tons [4].
Stem cankers and twig dieback are one of the most serious diseases of fig and loquat worldwide. The casual agents of these diseases belong to different families of ascomycetous fungi, including Diaporthaceae [5], Nectriaceae, Botryosphaeriaceae [6][7][8][9], and Ceratocystidaceae [10]. These pathogens cause almost indistinguishable symptoms. In a recent survey aimed to identify ascomycetous pathogens associated with a widespread decline of fruit orchards in southern Iran, Bionectriaceae isolates were the fungi recovered most frequently from fig and loquat trees with symptoms of stem cankers and twig dieback in Fars Province.
In this study, Bionectriaceae isolates, recovered from fig and loquat trees with symptoms of stem cankers and twig dieback in the Fars Province (southern Iran), were identified as a new species of Stilbocrea and their pathogenicity was evaluated to fulfill Koch's postulates and to determine if they were able to infect other fruit trees of economic importance in Iran. 'Siah,' and 'Bargchenary' among fresh figs, and 'Pouzdonbali' among caprifigs, as well as loquat trees, were sampled. Trunks, branches, and shoots with symptoms of canker, dieback, and decline were collected. Isolations were performed from transverse sections of symptomatic stems and twigs. Small tissue pieces (5 × 5 mm) from the margins between healthy and discolored wood were picked up with a scalpel, washed under running tap water, disinfected for 1 min in 70% ethanol, 1 min in a 2% sodium hypochlorite solution, and subsequently rinsed twice in sterile distilled water [8]. The disinfected segments were air-dried on a sterile paper towel for 10 min and then plated in Petri dishes containing potato dextrose agar (PDA, extract of 300 g/L boiled potato, 20 g glucose monohydrate, 15 g/L agar, distilled water) amended with tetracycline (1 mg/L). Plates were incubated at 25 • C for seven days. Emerging colonies were transferred into water agar (WA, 20 g/L agar, distilled water) and pure cultures were obtained by picking up single hypha tips after three days of incubation.

Morphological Characterization
All Bionectriaceae isolates were identified and characterized according to Voglmyr & Jaklitsch [16]. For the colony morphology characterization, mycelial plugs from each isolate were placed on PDA and malt extract agar (MEA, 20 g/L of malt extract, 15 g/L agar, distilled water) and incubated at 25 • C with approximately 12 h of daylight and 12 h of darkness. Identification of isolates was based on colony morphology, pigment production on PDA, microscopic structures including conidiophore morphology, phialide shape and type, and conidial shape and size. Fungal structures were mounted in lactic acid, and 30 conidia and other fungal structures were measured. Temperature-growth relationships and average growth rates were tested on PDA and MEA (80 mm Petri dishes with 25 mL media) measured after 14 days of incubation under 12-h photoperiod with three replicate dishes per isolate and incubated at 5,10,15,20,25,30, and 35 • C [16]. Isolates with the same morphological and cultural characteristics were grouped and representative isolates were further analyzed.

Phylogenetic Analyses
New sequences generated in this study were edited, proofread, and concatenated in BioEdit v. 7.0.9.0 [26]. They were blasted against the NCBI's GenBank nucleotide database to obtain highly similar sequences to representative isolates and preliminary identifications for phylogenetic inferences. The sequences generated in this study were aligned with the downloaded GenBank sequences by Clustal X [27] with subsequent visual adjustment. Partition homogeneity tests were conducted on combined nuclear gene alignment by PAUP* 4.0a136 [28] using 100 replicates and the heuristic general search option. Bayesian inference analyses on individual and concatenated ITS, rpb2, and tef1 loci were carried out to reconstruct the phylogenetic trees, using MrBayes 3.1 [29]. The phylogenetic analysis of the Bionectriaceae dataset included 35, 21, 16, and 15 ingroup taxa for individual gene ITS, rpb2, tef1, and their concatenated combination, respectively. Two Nectriaceae members, including Nectria cinnabarina (Tode) Fr. strain AR4477 and Thyronectria rhodochlora (Mont.) Seeler strain NP2 were used as outgroup taxa in all phylogenetic trees (Supplementary Table S1). Estimation of the best-fit nucleotide substitution model was determined by MrModelTest 2.3 [30]. Two independent Markov chain Monte Carlo (MCMC) runs 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. To conduct a phylogenetic comparison, maximum likelihood (ML) estimation was carried out using PHYLIP DNAML [31] with the same data set. Evaluation of the robustness of the maximum likelihood trees was conducted by the bootstrapping method with 1000 replicates. TreeGraph was used for editing and displaying phylogenetic trees [32]. Alignments and trees were submitted to TreeBASE [33].

Pathogenicity Tests
A preliminary pathogenicity evaluation of representative isolates was conducted on detached shoots (5-9 mm in diameter) collected from 5-to 12-year-old fig and loquat trees that were cut into pieces of 25-30 cm in length. A pathogenicity test was also carried out on mature one-year-old fig 'Sabz' (the most widely grown commercial cultivar) and loquat saplings in greenhouse conditions at 26 ± 3 • C. Saplings were inoculated in February 2020. To determine the potential host range of representative isolates, additional pathogenicity tests were performed on detached stems of other fruit trees, including almond, olive, apple, quince, and pomegranate, that were adjacent to the fig and loquat trees in surveyed orchards. In all pathogenicity tests, the stem surface was cleaned and disinfested with 70% ethanol. The inoculation site was wounded using a 6-mm sterilized cork borer to remove the bark. A 6-mm diameter disc was then taken from the margin of a seven-day-old culture on PDA, inserted into the wound, covered with the bark disk, and sealed with Parafilm ® (Bemis Packaging, Sheboygan Falls, WI, USA) to prevent contamination. Non-colonized PDA agar plugs were used for the negative controls [34]. Both inoculated and control shoots and stems were put into glass bottles containing 100 mL of sterilized water and kept under greenhouse conditions at 25 ± 2 • C. Experiments were arranged according to a completely randomized design. On detached shoots and stems, symptoms were recorded 21 days post-inoculation (dpi) by gently removing the bark and measuring the lesion length. Inoculated saplings and their respective controls were taken back to the laboratory 30 dpi, the bark was removed, and symptoms severity was rated by measuring the lesion length. For re-isolations from artificially inoculated stems and saplings, five pieces (2 × 5 mm) of necrotic tissue from the edge of each lesion were cut, surface-disinfected [8], and placed on PDA in an attempt to re-isolate the inoculated fungi and complete Koch's postulates. The isolates were identified based on morphological characteristics.  Figure 1A,E). The internal symptoms included brown to dark brown discoloration of wood that, in cross-sections, appeared as wedge-shaped necrosis ( Figure 1B,F).

Field Surveys and Disease Symptoms
From the sampling in Estahban, Darab, Firuzabad, Jahrom, Kazerun, Khafr, Neyriz, and Shiraz counties (Fars Province), 35 isolates out of a total of 355 recovered from symptomatic fig trees, and 8 out of 77 recovered from loquat trees, respectively, were identified as a Bionectriaceae taxon based on morphological and molecular characteristics ( Table 2).
Cardinal temperatures for growth: Minimum 15 • C, maximum 35 • C, optimum 30 • C. Colonies on PDA were cottony, aerial mycelium was white at first and then from dark herbago green [37] (p. 69) to olivaceous gray [37] (p. 121) at the center, olivaceous buff at the middle ring [37] (p. 69), and with edge white (Figure 6A,B). Slow-growing colonies on PDA, with a radial growth rate of 1.25-4.   Notes: This species belongs to the Stilbocrea clade of the phylogenetic tree of Bionectriaceae Voglmyr & Jaklitsch [16] in the proximity of S. walteri (Figures 2-5). Stilbocrea banihashemiana sp. nov. differentiates from S. walteri and S. macrostoma by its two different types of globose and allantoid conidia, branched, verticillioid, cylindrical to lageniform phialides and its unique sequences of nuclear genes. Isolates were recovered from various sites in Fars Province of Iran.

Pathogenicity Tests
In pathogenicity tests on detached shoots and one-year-old fig and loquat saplings, isolates of S. banihashemiana caused different types of symptoms such as cankers, bark flaking, wood discoloration, twig dieback, decline, and growth reduction. In particular, on loquat saplings, they induced dieback, decline, leaf yellowing, and defoliation ( Figure 7A,B). On detached stems of both fig and loquat, necrotic lesions developed upwards and downwards from the inoculation point ( Figure 8A-F). Internal symptoms consisted of discolored vascular tissues that became reddish-brown to dark brown and wedge-shaped, deep wood necroses visible in cross-sections of stems, eventually leading to cambium death ( Figure 7F). Both external and internal symptoms resulted in the rapid and progressive death of detached shoots at seven dpi. The primary symptoms in inoculated loquat saplings were wilting, defoliation, dieback, decline, and, finally, death of the whole sapling ( Figure 7A,B). On fig saplings, isolates of S. banihashemiana induced cankers at first ( Figure 7C) and nine months after inoculation, yellowing, and defoliation were also observed. Dieback and mortality were not observed on inoculated fig saplings (Figure 7D,E) up to 12 months after inoculation.
isolates of S. banihashemiana caused different types of symptoms such as cankers, bark flaking, wood discoloration, twig dieback, decline, and growth reduction. In particular, on loquat saplings, they induced dieback, decline, leaf yellowing, and defoliation ( Figure  7A,B). On detached stems of both fig and loquat, necrotic lesions developed upwards and downwards from the inoculation point ( Figure 8A-F). Internal symptoms consisted of discolored vascular tissues that became reddish-brown to dark brown and wedge-shaped, deep wood necroses visible in cross-sections of stems, eventually leading to cambium death ( Figure 7F). Both external and internal symptoms resulted in the rapid and progressive death of detached shoots at seven dpi. The primary symptoms in inoculated loquat saplings were wilting, defoliation, dieback, decline, and, finally, death of the whole sapling ( Figure 7A,B). On fig saplings, isolates of S. banihashemiana induced cankers at first ( Figure 7C) and nine months after inoculation, yellowing, and defoliation were also observed. Dieback and mortality were not observed on inoculated fig saplings (Figure 7D,E) up to 12 months after inoculation. In pathogenicity tests aimed at evaluating their potential host range, S. banihashemiana isolates were pathogenic to all tested fruit trees, including almond, olive, apple, and quince ( Figure 8G-N In pathogenicity tests aimed at evaluating their potential host range, S. banihashemiana isolates were pathogenic to all tested fruit trees, including almond, olive, apple, and quince ( Figure 8G-N), except pomegranate. Stilbocrea banihashemiana was re-isolated from infected symptomatic tissues of detached stems and saplings of fig and loquat, as well as branches of other artificially inoculated fruit trees.

Discussion
The widespread presence and high incidence of stem cankers and twig dieback associated with tree decline in fruit tree orchards in southern Iran, particularly on  [5,6,10,[38][39][40][41][42]. Some of these species, in particular L. theobromae and N. parvum, both in the Botryosphaeriaceae family, are cosmopolitan and very polyphagous pathogens, often reported as causal agents of perennial stem cankers on woody plants [41,43]. Moreover, members of Botryosphaeriaceae were reported to be responsible for limb cankers in loquat [7,8]. During the survey of orchards in southern Iran, fungi of Nectriaceae and Didymellaceae families were also recovered from declining fig and loquat trees, and most of them proved to be pathogenic [44]. However, this is the first report of a species of the Bionectriaceae family as the causal agent of trunk cankers and stem blights of fig and loquat. Morphological analyses and multiple gene phylogenetic analysis of nuclear loci (ITS, rpb2, and tef1) revealed all these isolates recovered from cankers and stems with symptoms of dieback belonged to a new species, which here has been described formally and named Stilbocrea banihashemiana sp. nov. Previous studies reported two other species of Stilbocrea, S. walteri and S. macrostoma, in Iran as pathogens of citrus and oak, respectively [17,18].
According to the phylogenetic analysis of three genes (ITS, rpb2, and tef1) and their concatenated combination, the S. banihashemiana sp. nov. isolates recovered from fig and loquat trees clustered in a monophyletic group in the Stilbocrea clade of Bionectriaceae phylogenetic tree [16]. Bayesian inference and maximum likelihood analyses showed that protein-coding genes (rpb2 and tef1), mostly tef1, have sufficient discriminatory power to differentiate Bionectriaceae members, particularly Stilbocrea species. The S. banihashemiana sp. nov. appeared to be a sister taxon of S. walteri in Bayesian and maximum likelihood trees but differed from S. walteri at 15, 55, and 81 variable nucleotide sites in the ITS region, rpb2, and tef1 genes, respectively. We observed a low diversity within this new lineage so that of the 2794 nucleotides characters included in the aligned three genetic regions (ITS, rpb2, and tef1), only nine variable nucleotide sites were detected.
Stilbocrea banihashemiana sp. nov. and S. walteri have some morphological characteristics in common, such as the lack of stilbella-like asexual structure and production of simple phialides in pure culture [16]. Conversely, other Stilbocrea species, e.g., S. colubrensis, and S. macrostoma produce synnemata in their asexual phase [15]. Stilbocrea banihashemiana sp. nov. differs from S. walteri and other Stilbocrea species by producing two morphologically distinct types of conidia, globose and cylindrical, which formed on short and long phialides, respectively. By contrast, S. walteri has only cylindrical conidia on simple short phialides. According to Voglmyr & Jaklitsch [16], the morphology of sexual and asexual reproductive structures is insufficient to distinguish different species within Bionectriaceae and Nectriaceae. Synnematous and stilbella-like asexual structures are also produced in the Nectriaceae, e.g., in Nectria pseudotrichia Berk. & M.A. Curtis [45], and acremonium-like structures (a structure in Acremonium species in Bionectriaceae) may be found in other unrelated families of the Sordariomycetes [46]. On the other hand, apart from Geosmithia and Clonostachys species that have been widely studied, for most Bionectriaceae taxa, no sequence data are available even for the ITS rDNA regions. Therefore, presently Bionectriaceae species identification is more challenging than for other taxonomic groups of ascomycetous fungi.
Pathogenicity evaluation of S. banihashemiana isolates fulfilled Koch's postulates indicating this species is able to cause trunk and branch cankers, limb blight, and wood discoloration in fig and twig dieback in loquat. It was recovered consistently from symptomatic trees and is one of the most common ascomycetous fungi responsible for fig and loquat tree decline in southern Iran. Consequently, the decline of fig and loquat trees observed in southern Iran may be regarded as a disease with a complex etiology and, in this respect, it is similar to grapevine trunk diseases (GTDs), one of the major concerns for viticulture worldwide [47,48]. During field surveys, we observed fig cankers showed four distinct facies that were tentatively designated as types A, B, C, and D, respectively. Type A cankers included trunk lesions with zonations and were demonstrated to be induced by infections of Diaporthe cinerascens (syn. Phomopsis cinerascens) [5]. Type B cankers, originating from the crown of the trees and developing upward, were mostly caused by Nectriaceae species (Z. Bolboli and R. Mostowfizadeh-Ghalamfarsa, unpublished data). Type C cankers were extended, sunken lesions on the trunks, like those shown in the Results section (see Figure 1C,D). Type D cankers were characterized by bark crackings as well as discolored and dead areas on the trunk and main branches, like the canker shown in the Results Seciton (see Figure 1A). This study indicates that S. banihashemiana sp. nov. isolates can induce types C and D cankers on fig trees. Moreover, the external facies of cankers correlated with differences in the type of internal wood discoloration in transverse sections. Transverse sections of types C and D cankers always showed a wedge-shaped internal wood discoloration, whereas in other types of cankers, necroses were crescentshaped, irregular-shaped, or disk-shaped. Should these differences be confirmed on a larger scale and under different environmental conditions, the type of canker could be used as a diagnostic criterion for the preliminary identification of the causal agents.
To our knowledge, this is the first report of a Stilbocrea species infecting fig and loquat worldwide. Consistently with our results, in a previous study, isolates of S. macrostoma, a closely related species, induced dieback symptoms on inoculated two-year-old oak trees [18]. In pathogenicity tests on heterologous host plants, S. banihashemiana isolates were also pathogenic to almond, olive, apple, and quince, suggesting very likely this new fungal species has a broader host range than just fig and loquat. The ability to infect heterologous hosts has epidemiological relevance as it implies the risk of both cross-infection between different plant species and accidental introduction of pathogen inoculum into commercial crops through alternate host plants, as already hypothesized for several other polyphagous fungus and oomycete plant pathogens [41,[49][50][51].
The survey of fig and loquat orchards in southern Iran during 2019-2021 revealed S. banihashemiana was commonly associated with symptoms of stem cankers, twig dieback, and internal wood discoloration leading to tree decline. In previous studies, a high incidence of tree decline incited by fungi infecting stems and twigs was imputed to several factors, including a local abundance of fungal inoculum and environmental conditions predisposing the tree to the infection, such as frost, drought, water, and heat stresses [41,[52][53][54]. Moreover, recently it has been highlighted a correlation between the presence of fungi associated with GTDs and the origin of propagation material, corroborating the hypothesis that fungi causing stem cankers and twig dieback in commercial orchards originate from the nursery and an endophytic lifestyle favors their spread through asymptomatic plants [41,[55][56][57][58][59]. All these epidemiological aspects would deserve to be further investigated. On the basis of current knowledge, a sustainable management strategy for tree decline should include measures aimed at both preventing or mitigating the effect of predisposing environmental factors and reducing the amount of inoculum in the orchard, such as proper management of the irrigation to avoid water stress, use of windbreaks to shelter the trees from winds and of shading nets to protect branches and limbs from sun scalds as well as pruning to remove withered twigs and branches.