Two Novel Lasiodiplodia Species from Blighted Stems of Acer truncatum and Cotinus coggygria in China

Simple Summary Lasiodiplodia species are plurivorous plant pathogens found worldwide, especially in tropical and subtropical regions, that result in fruit and root rot, die-back of branches and stem canker, etc. During the exploration of the fungal diversity of blighted stem samples collected in northern China, two new Lasiodiplodia species, L. acerina G.H. Qiao & W.T. Qin and L. cotini G.H. Qiao & W.T. Qin, were discovered based on integrated studies of phenotypic features, culture characteristics and molecular analyses. They were described and illustrated in detail. This work provided a better understanding of the biodiversity, phylogeny and established concepts of the genus Lasiodiplodia. Abstract The Lasiodiplodia are major pathogens or endophytes living on a wide range of plant hosts in tropical and subtropical regions, which can cause stem canker, shoot blight, and rotting of fruits and roots. During an exploration of the stem diseases on Acer truncatum and Cotinus coggygria in northern China, two novel species of Lasiodiplodia, L. acerina G.H. Qiao & W.T. Qin and L. cotini G.H. Qiao & W.T. Qin, were discovered based on integrated studies of the morphological characteristics and phylogenetic analyses of the internal transcribed spacer region (ITS), translation elongation factor 1-α (TEF1-α), beta-tubulin (TUB2) and RNA polymerase II subunit b genes (RPB2). Lasiodiplodia acerina is a sister taxon of L. henannica and distinguishable by smaller paraphysis and larger conidiomata. Lasiodiplodia cotini is closely related to L. citricola but differs in the sequence data and the size of paraphyses. Distinctions between the two novel species and their close relatives were compared and discussed in details. This study updates the knowledge of species diversity of the genus Lasiodiplodia. Furthermore, this is the first report of Lasiodiplodia associated with blighted stems of A. truncatum and C. coggygria in China.


Introduction
Lasiodiplodia, established in 1896, is a member of the family Botryosphaeriaceae [1]. Species in the genus Lasiodiplodia have been associated with different plant diseases including fruit and root rots, die-back of branches and stem cankers. The type species of Lasiodiplodia (L. theobromae) was regarded as one of the cosmopolitan, plurivorous pathogens mainly inhabiting tropical and subtropical regions [2,3].
The main morphological characteristics of Lasiodiplodia include hyaline, smooth, cylindrical to conical conidioenous cells, which produce subovoid to ellipsoid-ovoid conidia and the conidia are hyaline without septa or dark-brown with single septae [4]. Species in the genus Lasiodiplodia were mostly differentiated based on the characteristics of the conidia and paraphyses [5]. Some other morphological characteristics, such as annelations of conidiogenous cells, the dimensions and papillate nature of conidiomata, septate and pigmented conidia as well as the pycnidial paraphyses have been gradually used to recognize the Lasiodiplodia species, but to what extent these characteristics are phylogenetically significant warrants further investigation [6].
The Genealogical Concordance Phylogenetic Species Recognition (GCPSR) concept is widely used to delineate different fungal species. This approach relies on determining the concordance between multiple gene genealogies and delimiting species where the branches of multiple trees display congruence [7]. The widespread application of phylogenies based on ITS, TEF1-α, TUB2 and RPB2 genes promotes the accurate identification of species in the genus Lasiodiplodia, and more and more species have been successively introduced over the years; at present, more than 70 Lasiodiplodia species have been identified [8][9][10]. Among them, some species have been introduced almost entirely on the basis of DNA sequence phylogenies. Although the phylogenies were derived from the analysis of multiple loci, some species were introduced only on the basis of minor differences in only one locus, and some species cannot be clearly separated phylogenetically [11][12][13]. Several accepted Lasiodiplodia species (L. brasiliense, L. laeliocattleyae, L. missouriana, L. viticola) may be hybrids based on a detailed phylogenetic analyses of five loci from 19 Lasiodiplodia species [14].
To provide a better understanding of Lasiodiplodia species diversity in China, recent collections of the genus on Acer truncatum and Cotinus coggygria were examined. Two previously unrecognized Lasiodiplodia species were discovered based on integrated studies of phenotypic features, culture characteristics and phylogenetic analyses of the combined sequences of ITS, TEF1-α, TUB2 and RPB2. Detailed comparisons were made between the new taxa and their close relatives.

Isolates and Specimens
Cultures were isolated from the blighted stems of Cotinus coggygria and Acer Truncatum collected from Beijing, China, from 2018 to 2019. Stem segments (0.5 cm × 0.5 cm × 0.2 cm) were cut from the boundary of the lesion or dead tissues, surface sterilized subsequently and incubated on potato dextrose agar (PDA, peeled potatoes 200 g, glucose 20 g, agar 18 g, add water to 1 L) at 25 • C for fungal isolation [15]. Specimens, purified cultures and the ex-type strains were deposited in the culture collection of Institute of Plant Protection, Beijing Academy of Agriculture and Forestry Sciences.

Morphology and Growth Characterization
Morphological characterization of colonies, such as colony appearance, color and spore production were observed and recorded following the method of previous studies [5,11,16] on three media (PDA, malt extract agar (MEA, malt extract 20 g, agar 18 g, add water to 1 L) and synthetic nutrient-poor agar (SNA, monopotassium phosphate 1 g, potassium nitrate 1 g, Magnesium sulfate heptahydrate 0.5 g, potassium chloride 0.5 g, glucose 0.2 g, saccharose 0.2 g, agar 20 g, add water to 1 L)) with each isolate three replicates. Microscopic characteristics were recorded based on 20 paraphyses, 20 conidiogenous cells and 50 conidia on PDA at 25 • C in darkness. Photographs were taken from material mounted in lactic acid with Axiocam 506 color microscope (Carl Zeiss, Aalen, Germany) using Zeiss Imager Z2 software. The new species were established based on the guidelines outlined by Jeewon and Hyde [17].

Sequence Alignment and Phylogenetic Analyses
Sequences of the investigated Lasiodiplodia species excluding those of our two new species for phylogenetic analyses were obtained from the NCBI using Tbtools v. 1.09876 [21] ( Table 1). Sequences were assembled, aligned and manually adjusted with BioEdit v.7.2.5 [22]. To identify the phylogenetic positions of L. acerina and L. cotini, the combined sequences of ITS, TEF1-α, TUB2 and RPB2 for all strains were used for the phylogenetic analysis by methods of maximum parsimony (MP), maximum likelihood (ML) and MrBayes analyses (BI) with Diplodia mutila and D. seriata as outgroups. NEXUS files were generated with Clustal X 1.83 [23] in Phylosuit v.1.2.2 [24].  ML analyses with 1000 bootstrap replicates were conducted using raxmlGUI v. 2.06 [25]. The best-fit model of nucleotide substitution for each dataset was determined using Mod-elFinder [26]. Topological confidence of resulted trees was assessed by maximum likelihood bootstrap proportion (MLBP) with 1000 replicates.
MP trees were generated in PAUP v.4.0b [27], using the heuristic search function with tree bisection and reconstruction as branch swapping algorithms and 1000 random addition replicates. Gaps were treated as a fifth character and the characters were unordered and given equal weight. MAXTREES were set to 5000, branches of zero length were collapsed and all multiple, equally parsimonious trees were saved. Tree length (TL), consistency index (CI), retention index (RI), rescaled consistency index (RC) and homoplasy index (HI) were calculated. Topological confidence of resulting trees was tested by maximum parsimony bootstrap proportion (MPBP) with 1000 replications, each with 10 replicates of random addition of taxa. BI analysis was conducted by MrBayes v. 3.2.6 [28] with Markov Chain Monte Carlo algorithm. Nucleotide substitution models were determined by ModelFinder and GTR + I+G + F was estimated as the best-fit model. Two MCMC chains were run from random trees for 2,000,000 generations and sampled every 100 generations. The first 2500 trees were discarded as the burn-in phase of the analyses, and Bayesian inference posterior probability (BIPP) was determined from the remaining trees. Trees were visualized in FigTree v1.4.4.

Phylogenetic Analyses
The combined ITS, TEF1-α, TUB2 and RPB2 data set comprised 74 taxa with D. mutila and D. seriata as the outgroups. The MP dataset consisted of 1823 characters, of which 1358 characters were constant, 115 characters were parsimony informative and 366 variable characters were parsimony uninformative. A total of 284 most-parsimonious trees with the same topology were generated, one of them is shown in Figure 1     Among all the strains, 141 represented 76 Lasiodiplodia spp. clustered together with high support (MPBP/MLBP/BIPP = 100%/100%/1). Three isolates (JZBHD 1902(JZBHD , 1904(JZBHD and 1905 representing L. acerina and three isolates (JZBPG 1901(JZBPG , 1903(JZBPG and 1905 representing L. cotini clustered as distinct lineages from other Lasiodiplodia spp., with the support values MPBP/BIPP = 85%/0.84 and MPBP/MLBP/BIPP = 98%/100%/1, respectively. They showed a close phylogenetic relationship, respectively, with L. henanica and L. citricola.  Conidiomata were semi-immersed or superficial stromatic on PDA within 14 d, and were solitary, smooth, globose, dark grey to black, covered by dark gray mycelia without conspicuous ostioles and up to 2525 µm in diameter. Paraphyses were filiform, cylindrical, aseptate, thin-walled, hyaline, apex rounded, occasionally swollen at the base and unbranched, arising from the conidiogenous layer, extending above the level of developing conidia, and were up to 39.4 µm long and 3.0 µm wide. Conidiophores were reduced to conidiogenous cells. Conidiogenous cells were hyaline, holoblastic, smooth, discrete, thin-walled, and were cylindrical to ampulliform. Conidia were initially hyaline, ovoid to cylindrical, with a 1-µm-thick wall, (21. [6] and L. huangyanensis (82 µm) [9]. In addition, L. henanica had smaller conidiomata (520 µm) ( Table 2), and vacuoles in the conidia, which were also different from L. acerina [6].  Figure 3. Conidiomata were semi-immersed or superficial stromatic, produced on PDA within 14 d, solitary, smooth, globose, dark grey to black, covered by dark gray mycelia without a conspicuous ostiole, up to 415 µm in diameter. Paraphyses arise from the conidiogenous layer, filiform, extending above the level of developing conidia, up to 41.9 µm long and 2.6 µm wide, hyaline, cylindrical, aseptate, thin-walled, apex rounded, occasionally swollen at the base and unbranched. Conidiophores were reduced to conidiogenous cells. Conidiogenous cells were hyaline, cylindrical to ampulliform, holoblastic, discrete, thin-walled and smooth. Conidia were initially hyaline, ovoid to cylindrical, with a 1-µm-thick wall, mature conidia turned brown with a median septum and longitudinal striations and sometimes with one vacuole, (19. Culture characteristics: Aerial mycelia on PDA were abundant, smoke-grey to olivaceous-grey with the colonies dark black on the reverse side of the plate after 7 d. The colonies radius reached 45 mm on PDA after 24 h, and mycelia entirely covered the Botryosphaeria dothidea and Verticillum dahlia have been isolated from diseased leaves and stems of C. coggygria [36][37][38]; to our knowledge, this is the first report of Lasiodiplodia being associated with A. truncatum and C. coggygria.
Along with an increasing number of species recognized in the genus Lasiodiplodia, our understanding of the genus will become more sophisticated and intelligible through the integrated studies on morphology and phylogeny. Accumulations of our knowledge on Lasiodiplodia will provide useful information for establishing reasonable species concepts, and understand co-relations between morphology and sequence data in the future, which will lay further foundations for the scientific management of stem blight diseases and improvement in the landscape effect in the process of urban greening construction.

Conclusions
This study recognized two novel Lasiodiplodia species from blighted stems of A. truncatum and C. coggygria, which were the first reports of Lasiodiplodia associated with these two horticulture trees in China. The discovery provided a better understanding of the biodiversity and phylogeny of the genus Lasiodiplodia and is beneficial for future evaluation of the potential usages and functions of the new species.