Diversity of Plectosphaerella within aquatic plants from southwest China, with P. endophytica and P. sichuanensis spp. nov.

Abstract Members of Plectosphaerella inhabit different substrates, including plants, soil and insects, and most species are pathogens causing large losses in agriculture. During a survey of endophytic fungi in aquatic plants in southwest China, 112 strains of Plectosphaerella were isolated, representing two new species, P. endophyticasp. nov. and P. sichuanensissp. nov., as well as two known species, P. cucumerina and P. pauciseptata. The novel taxa are described and illustrated here using combined morphological and multi-locus phylogenetic (LSU-ITS-TEF-1α-TUB2) analyses. Our result revealed Plectosphaerella species inhabiting within aquatic plants in southwest China, and the separation frequency of each species was presented.

Species of Plectosphaerella are distributed in a variety of habitats and have wide geographic distribution (Carlucci et al. 2012;Zhang et al. 2019). Some species are pathogens on the fruit, root or collar in Cucurbitaceae plant, causing large losses of melon, pumpkin and zucchini crops (Alfaro-García et al. 1996;Toyozo et al. 2005;Raimondo and Carlucci 2018). Several species harm other plants such as tomato, pepper, bamboo, and asparagus. And the symptoms of hosts are decomposition, breakdown and death (Antignani et al. 2008;Carlucci et al. 2012;Arzanlou et al. 2013). Besides diseased plants, P. sinensis Lei Su & Y.C. Niu was reported as endophytic fungus without causing obvious symptoms (Su et al. 2017). In addition, most species causing plant diseases were also isolated from soil, except for P. oratosquillae (P.M. Duc et al.) A.J.L. Phillips et al. originating from arthropod Oratosquilla oratoria (Duc et al. 2009).
Plectosphaerella cucumerina is the most widely distributed species, thriving on more than nine plant genera: Arabidopsis, Cucumis, Galium, Hydrilla, Nicotiana, Pyrus, Solanum, Viola and Austropotamobius etc., and also occurring in soil and paper (Alderman and Polglase 1985;Palm et al. 1995;Smith-Kopperl et al. 1999;Domsch et al. 2007;. Meanwhile, this species has a wide geographic distribution, and it has been reported from England, Italy, Canada, the Netherlands, Egypt etc. Carlucci 2018, Giraldo et al. 2019). The second species with wide geographic distribution, P. plurivora A.J.L. Phillips et al., was found in Australia, the Netherlands, Germany, USA etc. Its hosts include Lolium, Solanum, Nicotiana, Asparagus and it also occurs in soil (Carlucci et al. 2012;. However, other species did not show obvious habitats diversity and wide geographic distribution.
During our investigation of endophytic fungal diversity of aquatic plants in southwest China, among 1697 acquired strains, 112 strains belonging to Plectosphaerella were isolated. Based on morphological characteristics and phylogenetic analysis, two known species, P. cucumerina and P. pauciseptata, were described, and two new species, P. endophytica and P. sichuanensis, were proposed and illustrated. The geographic distribution and habitat diversity of Plectosphaerella in this study were also discussed.

Isolates and Morphology
Samples were collected from Yunnan, Guizhou, Sichuan provinces, Chongqing and Tibet from 2014 to 2017. The dominant hosts are Myriophyllum, Potamogeton, Hydrilla and Hippuris. Samples were placed in plastic bags, labeled and transported to the laboratory. Each leaf and stem was cut into segments 30-40 mm in length and washed thoroughly with tap water, then a surface-disinfection was carried out according to Su et al. (2016). The segments were cut into smaller sections of about 5×5 mm under the aseptic operating table. Firstly, washed 30 s in sterile water and soaked 2 min in 0.5% hypochlorite solution, then 30 s in sterile water and 2 min in 75% ethanol, finally 30 s in sterile water. Each ten sections were randomly isolated on rose bengal agar (RBA, Guangdong Huankai Microbial Sci and Tech), the antibiotics chloramphenicol (0.1 g l -1 ) was added to restrain bacterial growth. When a fungus grew up from the segments, some hyphae were picked up and transferred to potato dextrose agar (PDA, 200 g potato, 20 g dextrose, 18 g agar, 1000 ml distilled water) plates for incubation at 28 °C. After 10 days, colonies were transferred to different plates, including corn meal agar (CMA, 20 g cornmeal, 18 g agar, 1000 ml distilled water) and oatmeal agar (OA, 30 g filtered oat flakes, 20 g agar, 1000 ml distilled water). Colony characteristics, growth speed and other macrostructure from PDA plates were observed after 10 days. Microscopic characteristics such as mycelium, conidiophores and conidia were examined and measured after 3 days on CMA using BX51 microscope (Olympus); the sterile water was used as a mounting medium.
Pure cultures were deposited in the Herbarium of the Laboratory for Conservation and Utilization of Bio resources, Yunnan University, Kunming, Yunnan, P.R. China (YMF, formerly Key Laboratory of Industrial Microbiology and Fermentation Technology of Yunnan) and at the China Center for Type Culture Collection (CCTCC).

DNA extraction, PCR amplification and sequencing
Actively growing mycelium was scraped off from the surface of the culture and transferred to 2 ml Eppendorf micro-centrifuge tubes. Total genomic DNA was extracted follow the protocol of Guo et al. (2000). The internal transcribed spacer (ITS) and the 28S large subunit nuclear ribosomal RNA (LSU rRNA) were amplified using the primer pairs ITS1/ITS4 (White et al. 1990) and LROR/LR5 (Vilgalys and Hester 1990), respectively. Translation elongation factor 1-alpha (TEF-1α) and partial β-tubulin (TUB2) were amplified using the primer pairs EF-1251R/EF-688F (Alves et al. 2008) and Bt2a/Bt2b (Glass and Donaldson 1995), respectively. The PCR amplifications were conducted in 25 µl final volumes which consisted of 1.0 µl DNA template, 1.0 µl of each forward and reverse primers, 12.5 µl 2 × Master Mix and 9.5 µl ddH 2 O. The PCR reaction cycles were as follows: initial denaturation at 94 °C for 3 min; followed by 35 cycles of denaturation at 94 °C for 40 sec; the annealing extension dependent on the amplified loci (48 °C for LSU, 54 °C for ITS, 55 °C for TEF-1α and 58 °C for TUB2) for 1 min and extension at 72 °C for 2 min; a final extension at 72 °C for 10 min. PCR products were sequenced by TSINGKE Biological Technology in Kunming, China. The sequences are deposited in GenBank database and the accession numbers are listed in Table 1.

Phylogenetic analyses
The ITS sequences generated in this study were used as a query to search similar DNA sequences using BLASTn. All published DNA sequences were obtained from the Gen-Bank from relevant studies (Su et al. 2017;Phookamsak et al. 2019;Zhang et al. 2019), Monilochaetes infuscans Harter (Australiascaceae) was selected as an outgroup. The generated sequences were manually aligned with CLUSTAL_X v. 1.83 (Thompson et al. 1997) with default parameters. Aligned sequences of multiple loci were concatenated and manually adjusted through BioEdit version v. 7.0.4.1 (Hall 1999), and ambiguously aligned regions were excluded. The combined sequence was converted to a NEXUS file using MEGA6 (Tamura 2013). The alignment was deposited at Tree-Base http://purl.org/phylo/treebase/phylows/study/TB2:S27363.
Maximum-likelihood (ML) analysis was conducted by using RAxML (Stamatakis 2006) with the PHY files generated with CLUSTAL_X v. 1.83 (Thompson et al. 1997), using the GTR+GAMMA model. ML bootstrap proportions (MLBPs) were computed with 1000 replicates. Bayesian inference (BI) analysis was executed with MrBayes v. 3.2.2 (Ronquist and Huelsenbeck 2003). The Akaike information criterion (AIC) implemented in jModelTest version 2.0 was used to select the best fit models after likelihood score calculations (Posada 2008). HKY+I+G was estimated as the best-fit model under the output strategy of AIC, Lsetnst=6, rates=gamma. A Markov Chain Monte Carlo (MCMC) algorithm was used to generate phylogenetic trees with Bayesian probabilities. Two runs were executed simultaneously for 6,000,000 generations and sampled every 500 th generations, four chains containing one cold and three heated were run until the average standard deviation of the split frequencies dropped below 0.01, the stationarity of the analyses was confirmed in line with standards described by Sun and Guo (2010). The initial 25% of the generations of MCMC sampling were discarded as burn-in. The refinement of the phylogenetic tree was used for estimating BI posterior probability (BIPP) values. The tree was viewed in FigTree version 1.4 (Rambaut 2012).

Phylogenetic analyses
A total of 112 strains were identified as members of Plectosphaerella according to the BLASTn search results using the ITS sequences. At first, we carried out individual Strains and sequences generated in this study are emphasized in bold face. T ex-type cultures.
phylogenetic analyses with ITS sequences to resolve the taxonomic position of our strains using the sequences of the accepted species into Plectosphaerella. This tree is shown in Figure 1.  other strains formed two individual groups. Therefore, three representative strains of P. pauciseptata, a representative strain of P. cucumerina, and three representative strains of two novel taxa were chosen among the 112 strains for single-gene and combined phylogenetic analysis.
The four Bayesian trees derived from the single-gene sequence alignments (LSU, ITS, TEF-1α, TUB2) confirmed that the novel taxa were distant from other known species in Plectosphaerella. The Bayesian trees are available in the Suppl. material 1. The resulting combined sequence matrix included 2136 nucleotide positions (804 from LSU, 545 from ITS, 413 from TEF-1α, 374 from TUB2), with M. infuscans CBS 379.77 as the outgroup. The tree topology is shown in Figure 2, with the Bayesian posterior probabilities over 80% and ML bootstrap support over 50% indicated for respective clades. In this phylogenetic tree, P. endophytica represented by strain YMF 1.04701 was close to P. oratosquillae (NJM 0662 and NJM 0665) and formed a single clade with 0.97 Bayesian posterior probability and 57% ML bootstrap proportions. Similarly, P. sichuanensis represented by strains YMF 1.05081 and YMF 1.05082 were close to P. populi Ullah et al. (CBS 139623) with 1.00 Bayesian posterior probability and 100% ML bootstrap proportions. Considering distinct morphological differences, we propose to describe our isolates as two new species in Plectosphaerella.  Etymology. Latin, endophytica meaning endophytic, growing within plant tissue.
Culture characteristics. Colonies on OA reaching 52 mm diameter, on PDA reaching 48 mm diameter and on CMA reaching 43 mm diameter in 14 d at 25 °C. On PDA, colonies white, dense, fluffy hyphae growth in the medium surface, outermost mycelia formed an annule, margin smooth and entire, sporulation abundant, reverse pale yellow to white.
Culture characteristics. Colonies on OA reaching 50 mm diameter, on PDA reaching 47 mm diameter and on CMA reaching 42 mm diameter in 14 d at 25 °C. On PDA, colonies pale brown to white, flat, repressed, plicated, partly immersed, a few white aerial hyphae grew in the middle of the medium, margin regular, frontier distinct, reverse pale brown to white.

Discussion
Sexual reproduction of Plectosphaerella has only been reported for three species Phookamsak et al. 2019). Most strains, by contrast, show asexual morphs in substrate, so the main distinguishing characteristics of this genus are based on the ratio of septate conidia, conidial dimensions and shape, absence or presence of chlamydospores (Carlucci et al. 2012;Arzanlou et al. 2013). These simple features and similar lifestyle make identification more difficult, so the classical way is no longer a valid and reliable marker (Palm et al. 1995;Zare et al. 2007;Carlucci et al. 2012). The ITS and LSU regions were considered to be a necessary condition for species identification of Plectosphaerella (Duc et al. 2009;Carlucci et al. 2012;Liu et al. 2013;Crous et al. 2015). However, Plectosphaerella species exhibit a relatively low degree of ITS or LSU molecular diversity, showing more molecular markers are needed to distinguish species (Carlucci et al. 2012). Recently, fragments of several protein-coding genes were selected to efficiently elucidate the taxonomy of this genus, including CaM, TEF-1α, TUB2 and RPB2 (Su et al. 2017;Phookamsak et al. 2019;Zhang et al. 2019). In this study, a multi-locus analysis improved the diagnostic level of the whole genus.
In this study, among 129 sample sites in Yunnan and Sichuan province, Plectosphaerella species were isolated from 13 sampling sites. Regions are arranged in order of isolation frequency: Eehai (39.29%), Dian Lake (20.54%), Fuxian Lake (13.39%), Daofu (8.04%) and other places were below 5%. It seems that distribution of Plectosphaerella is impacted by human activities, because there are more human activities around the first three sites. In addition, Plectosphaerella species only occur in six aquatic plants including Batrachium, Halerpestes, Hippuris, Hydrilla, Myriophyllum, and Potamogeton, although we investigated 30 aquatic plants. So Plectosphaerella species exhibit medium host diversity and relatively narrow geographic distribution when aquatic plants serve as hosts.
Secondly, the separation frequency of Plectosphaerella pauciseptata was the highest at 68.78%, followed by P. cucumerina at 24.11%, and two new species were the lowest at 3.57%. According to this data and previous studies, P. cucumerina and P. pauciseptata are still the most widely distributed species either on land or within aquatic plants, although P. pauciseptata is more common in aquatic plants. On the other hand, only four species were identified among 112 strains. One reason may be that species diversity of Plectosphaerella is low within aquatic plants. Other reasons may be Plectosphaerella species exhibit a relatively low degree of ITS molecular diversity. Based on this, perhaps we treat some different species as two known species, which reduce the diversity of Plectosphaerella. Regrettably, we did not preserve all strains except for representative strains. Or else, we should sequence other loci of all strains, especially for two species, P. pauciseptata, P. cucumerina, more loci are needed to distinguish them.
This study revealed Plectosphaerella species within aquatic plants from the southwest of China, although only 112 strains and four species were reported, which also enrich the ecology and diversity of the genus. However, our survey was limited, hence it is necessary to know if our result is concordant with those of other regions. So in-depth work is required to obtain a more integrated knowledge of their biodiversity and distribution in water bodies.