Additions to The Knowledge of Tubakia (Tubakiaceae, Diaporthales) in China

The species of Tubakia (Tubakiaceae, Diaporthales, Sordariomycetes) have often been reported as endophytes and pathogens on woody plants. During the investigation of Tubakia species from Fagaceae trees in China, 46 isolates were obtained from diseased leaves and seeds. The characterization of these isolates was based on the observation of morphological characters, the effect of temperature on mycelial growth rate, as well as the combined genes of ITS, tef1 and tub2. As a result, six species were identified: Tubakia americana, T. cyclobalanopsidis sp. nov., T. dryinoides, T. koreana, T. paradryinoides and T. quercicola sp. nov. Among these, T. koreana and T. paradryinoides were firstly discovered in China. Pathogenicity tests were conducted using the conidial suspension on young, excised leaves for these six species, which showed that they were mildly pathogenic to four Fagacece hosts: C. mollissima, Q. acutissima, Q. aliena var. acutiserrata and Q. variabilis.

Saccardo introduced the genus Actinopelte with A. japonica as the type [2].Subsequently, Höhnel added A. americana and A. dryina to this genus [6].Yokoyama and Tubaki described A. castanopsidis, A. rubra and A. subglobosa according to comprehensive examinations based on Japanese collections [7].Since Actinopelte turned out to be illegitimate (later homonym of Actinopelte Stitzenb.1861), Sutton introduced the alternative name Tubakia [1][2][3].Several species were revealed from leaves of Quercus spp. in the USA, namely T. hallii, T. macnabbii and T. tiffanyae [3].Braun revised this genus based on morphological and phylogenetic data, and Tubakia was expanded as a family Tubakiaceae [1].Subsequently, five additional species named T. californica, T. melnikiana, T. oblongispora, T. paradryinoides and T. sierrafriensis were introduced based on in vivo and in vitro morphological analyses, as well as phylogenetic data [1].In addition, T. koreana and T. lushanensis were described from China and Korea, respectively [8,9].Until now, a total of 19 species have been accepted into this genus.
In Japan, Matsumura et al. examined the endophyte communities of seven evergreen Quercus species, showing that host identity and ecology were significantly associated with Tubakia community structure [20].T. iowensis as a serious pathogen causes necrosis of the leaf tissue along the veins and the eventual death of entire leaves on Q. macrocarpa in North America [11,12].T. dryina was discovered as leaf pathogens of Fagus sylvatica, Quercus robur and Tilia cordata in Poland [10].
In China, six species of Tubakia have been reported, viz.T. americana on seeds of Quercus variabilis [16], T. chinensis on Castanea henryi [15], T. dryina from Quercus spp.and Castanea spp.[13], T. japonica on Castanea mollissima [18], T. lushanensis from leaves of Quercus palustris [9] and T. seoraksanensis on Quercus mongolica [17].The present study describes two novel species and four known species of Tubakia in China based on both morphology and phylogeny.

Isolation and Morphological Characterization
From 2018 to 2020, specimens were collected during investigations for plant diseases in Mount Huang (Huangshan City), Shushan Forest Park (Hefei City) and Zipeng Mountain (Hefei City) of Anhui Province; Guangzhou City of Guangdong Province; Kuankuoshui national nature reserve (Zunyi City) of Guizhou Province; Kikunshan National Nature Reserve (Xinyang City) and Yaoshan Mountain (Pingdingshan City) of Henan Province; Foping County (Hanzhong City), Panjiawan Forest Park (Baoji City) and Zhuque National Forest Park (Xian City) of Shaanxi province.Isolates of Tubakia in this study were obtained from diseased leaves and seeds of C. mollissima, Q. acutissima, Q. aliena var.acuteserrata, Q. glauca and Q. variabilis.
The leaf and seed samples were first surface-sterilised for 1 min in 75% ethanol, 3 min in 1.25% sodium hypochlorite and 1 min in 75% ethanol, rinsed for 2 min in distilled water and blotted on dry sterile filter paper.Then, the diseased areas of the leaves were cut into 0.5 × 0.5 cm pieces using an aseptic razor blade, transferred on to the surface of potato dextrose agar plates (PDA; 200 g potatoes, 20 g dextrose, 20 g agar per litre) and incubated at 25 °C to obtain pure cultures.The cultures were deposited in the China Forestry Culture Collection Center (CFCC; http://cfcc.caf.ac.cn/) and the specimen was deposited in the Herbarium of the Chinese Academy of Forestry (CAF; http://museum.caf.ac.cn/).
To determine the effect of temperature on mycelial growth and the optimal growth temperature, the representative isolates were cultured on PDA and malt extract agar (MEA, 30 g malt extract, 5 g mycological peptone, 15 g agar per litre) for further assays.After seven days of incubation at 25 °C, 5 mm diam.mycelial plugs were transferred from the edge of the colonies to the center Petri plates.The plates were incubated at 5-10-15-20-25-30-35-40 °C in the dark.Three Petri plates were used for each temperature as replicates.
Microscopic structures of the fungus growing on a medium were mounted in water and examined under an Axio Imager 2 microscope (Zeiss, Oberkochen, Germany).At least 30 measurements were made for each structure examined.

DNA Extraction, Amplification and Sequencing
Genomic DNA was extracted from the fresh mycelium harvested from PDA plates after 4 days using a cetyltrimethylammonium bromide (CTAB) method [22].For initial species confirmation, the internal transcribed spacer (ITS) region was sequenced for all isolates.The BLAST tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi,15 August 2022) was used to compare the resulting sequences with those in GenBank (Table A1).After confirmation of Tubakia species, two additional gene regions coding for translation elongation factor 1-alpha (tef1), and beta-tubulin (tub2) were sequenced.Three loci were amplified with the following primer pairs, ITS1 and ITS4 for ITS [23], T1 and 688F for tef1 [24], and T1/Bt2a and Bt2b for tub2 [25,26].The primer pairs and amplification conditions for each of the above-mentioned gene regions are provided in Table 1.
A PCR reaction was conducted in a 20 µL reaction volume, and the components were as follows: 1 µL DNA template (20 ng/µL), 1 µL forward 10 µM primer, 1 µL reverse 10 µM primer, 10 µL T5 Super PCR Mix (containing Taq polymerase, dNTP and Mg 2+ , Beijing Tisingke Biotech Co., Ltd., Beijing, China), and 7 µL sterile water.Amplifications were performed using a T100 Thermal Cycler (Bio-Rad, Hercules, CA, USA).Strands were sequenced in both directions using PCR primers.All amplified PCR products were estimated visually with 1.4% agarose gels stained with ethidium bromide and then PCR positive products were sent to Sangon Biotech (Shanghai) Co., Ltd., (Beijing, China) for sequencing.The new sequences generated in this study, as well as the reference sequences of all isolates used in the present study, are listed in Table 2.

Phylogeny
The sequences generated in this study were supplemented with additional sequences obtained from GenBank (Table 2).The dataset consisted of 94 sequences, including one outgroup taxon, Melanconis groenlandica (CBS 116540).The sequences were aligned with the MAFFT v.7, after which the alignments were manually corrected using MEGA v.7.0.[27,28].Phylogenetic analyses, including Maximum Likelihood (ML) and Bayesian Inference (BI) methods, were conducted for the single gene sequence data sets of the ITS, tef1 and tub2, and the combined data set of all three gene regions.ML analyses were conducted using RAxML-HPC BlackBox 8.2.10 on the CIPRES Science Gateway portal (https://www.phylo.org,12 June 2022), employing a GTRGAMMA substitution model with 1000 bootstrap replicates [29,30].Bayes inference was conducted using a Markov Chain Monte Carlo (MCMC) algorithm in MrBayes v.3.0 [31].Two MCMC chains were run from a random starting tree for 1,000,000 generations, resulting in a total of 10,000 trees.The first 25% of trees sampled were discarded as burn-in, and the remaining trees were used to calculate the posterior probabilities.Branches with significant Bayesian Posterior Probabilities (BPP > 0.9) were estimated in the remaining 7,500 trees.Phylogenetic trees were viewed with FigTree v.1.3.1 and processed by Adobe Illustrator CS5.

Pathogenicity Test
The isolates of each Tubakia species were used for a pathogenicity test on four hosts, viz.C. mollissima, Q. acutissima, Q. aliena var.acuteserrata and Q. variabilis.Each isolate was incubated on PDA for 7-15 days at 25 °C to achieve spore suspension.Fresh leaves without visible diseases were collected from 1-year-old Fagaceae plants and used for the tests.These leaves were surface sterilized in 75% ethanol for 3 min and 1% sodium hypochlorite for 3 min, and then rinsed thrice in sterile distilled water.
After washing and air drying, these leaves (six of each species) were surface wounded by a sterile needle, and 10 µL of conidial suspension (1 × 10 6 conidia/mL) was inoculated on the wounds.Sterile water was used as a control treatment.Inoculated leaves were placed in glass containers on top of moist paper and sealed.The containers were placed in a growth chamber and incubated at 25 °C with an alternation of 12 h of light and 12 h of darkness for 14 days.Symptom development of the leaves was checked daily and recorded for up to 14 days.

Statistical Analyses
Regression analyses were applied to the means of all independent quantitative variables.Nonlinear regression models were evaluated for describing the relationship between mycelial growth and temperature.The Gram-Charlier A series (GCAS) was selected because it provided a good fit for all isolates.All these analyses were conducted using OriginPro 2018 [32].GCAS were fitted to the values of mycelial growth versus temperature for each isolate, and the optimum temperatures were calculated in the fitted equations.

Phylogenetic Analyses
The combined three-gene sequence dataset (ITS, tef1 and tub2) was analysed to determine the phylogenetic position of the new isolates obtained in this study.A total of 1919 characters, including gaps (686 for ITS, 665 for tef1 and 568 for tub2), were included in the dataset used in the phylogenetic analyses.Of these characters, 1024 were constant, 218 were variable, but parsimony-uninformative and 677 were parsimony-informative.The best ML tree (lnL =−13482.15)revealed by RAxML is shown as a phylogram in Figure 1.The topologies resulting from ML and BI analyses were congruent (Figure 1).Isolates in the present study were separated into seven supported clades in

Morphology
After seven days of incubation, no mycelial growth was observed at 40 °C.Tubakia quercicola grows faster than the other species (0.73 cm/day) on PDA at 25 °C, whereas Tubakia cyclobalanopsidis grows the slowest (0.54 cm/day).Furthermore, the rate of colonies growth on PDA is faster than those on MEA.The growth ranges and growth rate of each temperature are significant between T. americana clade I and T. americana clade II.All results of the effects of temperature on mycelial growth rate are shown in Table 3 and Fig
Culture Characteristics: Tubakia americana clade I: Colonies on PDA incubated at 25 °C in the dark with an average radial growth rate of 9-10 mm/d and occupying an entire 90 mm Petri dish in 14 d, dark green on the bottom, aerial mycelium cottony, white initially, then becoming off-white.Colonies with optimal growth at 25 °C on MEA, attaining a diameter of 45-60 mm after 7 days, initially with a distinct ring of sparse aerial mycelium, later developing concentric rings of white to yellow aerial mycelium with wet conidial masses that are initially hyaline, becoming creamy white then faintly yellow, coalescing into large areas.Tubakia americana clade II: Colonies on PDA incubated at 25 °C in the dark with an average radial growth rate of 6-7 mm/d and occupying an entire 90 mm petri dish in 10 d.When young, yellow green mycelium mostly immersed; when old, in the middle dark green, with dark green parts covered with continuously growing white mycelia, dark green-to-black in reverse.Cultures incubated on MEA at 25 °C in darkness, attaining 29 Host Range and Distribution: On Quercus (acutissima, aliena var.acuteserrata, bicolor, coccinea, glauca, macrocarpa, robur, rubra, variabilis), Fagaceae, China (Anhui Province, Guangdong Province, Henan Province and Shaanxi Province), North America (USA, Illinois, Iowa, Missouri, Wisconsin).
Notes: In this study, 12 isolates were obtained from diseased leaves of Q. acutissima, Q. aliena var.acuteserrata and Q. variabilis, as well as the rotted seeds of Q. glauca.These isolates were separated into two clades within the species Tubakia americana based on branch length (Figure 1).Etymology: Named after the original genus name of the host Quercus glauca, Cyclobalanopsis.
Culture Characteristics: Colonies on PDA incubated at 25 °C in the dark with an average radial growth rate of 7-9 mm/d and reaching 52-57 mm diam. in 7 d.When young, round, dark green in the center and white at the edge, with some dark green parts covered with continuously growing mycelia.When old, tight, dark green and white at the edge, with dark green parts covered with continuously growing white mycelia.Cultures were incubated on MEA at 25 °C in darkness, attaining 37-41 mm diam.after 7 d (growth rate 5-6 mm diam./d),creamy white to faintly yellow with regular margin, white near the centre and hyphae clusters, reverse faintly yellow to yellow rings.
Culture Characteristics: Colonies on PDA incubated at 25 °C in the dark with an average radial growth rate of 8-9 mm/d and occupying an entire 90 mm petri dish in 10 d; white initially, aerial mycelium cottony, then becoming moist yellow green, covered with grayish white mycelium.Cultures incubated on MEA at 25 °C in darkness, attaining 61-74 mm diam after 10 days, margin scalloped, creamy white initially, then with fluffy pale brown to dark brown mycelia, yellow, pale brown to brown in reverse, with a cream white edge.
Notes: The holotype of T. dryinoides was collected from leaves of Q. phillyraeoides.In this study, isolates collected from diseased leaves of Q. acutissima, Q. aliena var.acuteserrata and seeds of Q. glauca, which formed a well-supported clade with the ex-type strain MUCC2292 (Figure 1).Culture Characteristics: Colonies on PDA at 25 °C for 10 days attain 78-90 mm in diameter.When young, round, cream white in the center, with some moist mycelium; when old, hyphae lush, gray to ash black, white at the edge.On MEA with optimal growth at 25 °C, attaining 43-50 mm after 7 days, margin scalloped, faintly yellow in the centre and with a cream white edge, they have wrinkles, yellow to brown in reverse, white at the edge.
Host range and distribution: on Quercus (acutissima and glauca), Fagaceae, China (Anhui Province) and Japan.
Culture Characteristics: Colonies on PDA were incubated at 25 °C in the dark with an average radial growth rate of 9-11 mm/d and occupying an entire 90 mm Petri dish in 14 d, dark green on the bottom, aerial mycelium cottony, white initially, then becoming greyish.Optimal growth at 25 °C on MEA in darkness, colonies attaining 39-45 mm after 7 days, dingy white to pale yellow with regular margin, becoming yellowish gray with concentric rings in reverse, conidial formation not observed.The colony growth rate on MEA reached 6 mm/day, which is a growth that is slower than on PDA.Host range and distribution: on Quercus aliena var.acuteserrata, Fagaceae, China (Guizhou Province and Shaanxi Province).
Notes: The phylogenetic analysis of a combined three genes alignment (ITS, tef1 and tub2) showed that T. quercicola clustered into a well-supported clade.Morphologically, T. quercicola is similar to T. dryina in conidial size [1].However, T. quercicola can be distinguished from T. dryina by sequence data (5/631 in ITS; 25/604 in tef1 and 20/535 in tub2).Furthermore, the MEA's colony colour of T. quercicola is different from T. dryina (surface: creamy white to faintly yellow vs. creamy white, dark grey, yellow to medium brown) [1].

Pathogenicity Test
The results of the pathogenicity test on four hosts are shown in Table 4.We can see that the aggressiveness of the tested species for different leaves differed significantly.For instance, T. paradryinoides was not pathogenic to Q. aliena var.acutiserrata, but the other fungal species could cause lesions on all tested host leaves.In addition, T. paradryinoides had a high infection rate in C. mollissima, Q. acutissima and Q. variabilis, but was not pathogenic to Q. aliena var.acutiserrata.

Discussion
It is well known that Tubakia species have a wide geographic distribution mainly inhabiting Fagaceae hosts.Tubakia may appear on leaf or twig tissues simultaneously with other agents.Species of Tubakia may have an endophytic phase of growth [19,[33][34][35].Some endophytic Tubakia species have mutualistic interactions with their plant hosts, including the concept of a sentinel tree [36].Currently, the majority of Tubakia species are mainly endophytes.However, T. iowensis cause a serious leaf disease (bur oak blight), and T. hallii and T. macnabbii have also been related to the significant defoliation of Quercus spp.[11,12].
In this study, we investigated the diversity of pathogens in Fagaceae plants in China and obtained 46 isolates belonging to Tubakia.Based on morphology and a concatenated three-gene phylogenetic analysis, the isolates were assigned to six species (viz.T. americana, T. cyclobalanopsidis, T. dryinoides, T. koreana, T. paradryinoides and T. quercicola).Each species formed a well-supported monophyletic clade in the phylogenetic analysis.Since the inception of Tubakia in 1913, its delimitation has undergone several changes.Twentyone species are phylogenetically recognized in Tubakia, including T. cyclobalanopsidis and T. quercicola spp.nov.from the present study [1,5,8,9].
Tubakia cyclobalanopsidis sp.nov.appeared to be a sister species of T. paradryinoides in the phylogram (Figure 1), but differed from T. paradryinoides by 10, 21 and 17 variable nucleotide sites in the ITS, tef1 and tub2 genes, respectively.Morphologically, T. cyclobalanopsidis differs from T. paradryinoides by producing smaller conidia.Tubakia quercicola, newly discovered in this study, is a remarkable species in Tubakia.Morphologically, T. quercicola can be easily identified as a member of T. koreana or T. melnikiana, due to the size of conidiogenous cells and conidia.However, phylogenetic analyses demonstrated that it is a new clade (Figure 1).Bayesian inference and maximum likelihood analyses showed that protein-coding genes (tef1 and tub2), mostly tef1, have sufficient discriminatory power to differentiate T. quercicola.
The results revealed two clades within T. americana.The independence of T. americana clade I as a distinct clade is mainly supported by its unique tef1 sequence, which influences its position in the phylogenetic tree.Morphologically, all isolates in the two clades share the typical characteristics of T. americana [1,3].T. dryinoides was firstly discovered on Q. acutissima, Q. aliena var.acutiserrata and Q. glauca.T. koreana was firstly described on C. mollissima, Q. glauca and Q. variabilis.T. paradryinoides was firstly described on Q. glauca.Therefore, this study expands the habitat and host of T. americana, T. dryinoides, T. koreana and T. paradryinoides in China.
Pathogenicity tests of the six species identified in the present study were conducted on four host plants, which shows that T. quercicola had the highest incidence (Table 4).Furthermore, all tested Tubakia species showed significantly different lesion diameters on leaves (Table 4).Therefore, our studies revealed a broad diversity in pathogenicity and aggressiveness among six Tubakia species.
The proper identification of fungal species is necessary in disease control [37][38][39].Our knowledge of fungi and their relationships with plant hosts has increased exponentially due to the progress in bioinformatics and molecular phylogenetics.Tubakia species are endophytes in leaves and twigs of many species, but can also cause conspicuous leaf symptoms as plant pathogens.Therefore, identification of Tubakia species associated with hosts, as well as their lifestyles, is important.This study conducted a large-scale investigation of Tubakia associated with Fagaceae in China and provides morphological, molecular, and biological characterizations of these Tubakia species.This study not only enhances our understanding of the diversity of Tubakia species associated with Fagaceae, but also enriches knowledge of the host diversity of Tubakia species.

Conclusions
Six Tubakia species are identified from fagaceous hosts in China based on morphology and phylogeny; viz.T. americana, T. cyclobalanopsidis, T. dryinoides, T. koreana, T. paradryinoides and T. quercicola.This study enriches the species diversity of the genus, which will also promote its taxonomy and phylogeny.

Figure 1 .
Figure 1.A phylogram of Tubakia resulting from a maximum likelihood analysis based on a combined matrix of ITS, tef1 and tub2.Numbers above the branches indicate ML bootstrap values (left, ML BS ≥ 50%) and Bayesian Posterior Probabilities (right, BPP ≥ 0.9).The tree is rooted with Melanconis groenlandica (CBS 116540).Isolates from the present study are marked in blue and bold face.

Figure 2 .
Figure 2. The effect of temperature on the mycelial growth rate of six Tubakia species on PDA after seven days of incubation.

Figure 3 .
Figure 3.The effect of temperature on the mycelial growth rate of six Tubakia species on MEA after seven days of incubation.

Figure 4 .
Figure 4.The effect of temperature on the mycelial growth rate of six Tubakia species on PDA.The averages of radial growth rate and temperatures were adjusted to a nonlinear regression curve through the Analytis GCAS model.Data points are the means of two independent experiments of three replicated Petri dishes.The formula of the nonlinear fitting curve is as follows: T. americana

Table 1 .
Loci used in this study with PCR primers, and the process.

Table 2 .
Strains and GenBank accession numbers used in this study.

Table 3 .
An overview of colony diameters at various temperatures.

Table 4 .
Pathogenicity testing for six Tubakia species.