Fourteen New Species of Foliar Colletotrichum Associated with the Invasive Plant Ageratina adenophora and Surrounding Crops

Ageratina adenophora is one of the most invasive weeds in China. Following an outbreak in Yunnan in the 1960s, A. adenophora has been spreading in Southwest China at tremendous speed. Previous research indicated A. adenophora contained many Colletotrichum species as endophytes. In this study, we investigated the diversity of Colletotrichum in healthy and diseased leaves of the invasive plant A. adenophora and several surrounding crops in Yunnan, Guangxi, and Guizhou provinces in China, and obtained over 1000 Colletotrichum strains. After preliminary delimitation using the internal transcribed spacer region (ITS) sequences, 44 representative strains were selected for further study. Their phylogenetic positions were determined by phylogenetic analyses using combined sequences of ITS, actin (ACT), chitin synthase (CHS-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and beta-tubulin (TUB2). Combined with morphological characteristics, 14 new Colletotrichum species were named as C. adenophorae, C. analogum, C. cangyuanense, C. dimorphum, C. gracile, C. nanhuaense, C. nullisetosum, C. oblongisporum, C. parvisporum, C. robustum, C. simulanticitri, C. speciosum, C. subhenanense, and C. yunajiangense.


Introduction
Foliar fungal endophytes are defined as fungi that can colonize the internal plant leaf structure without causing apparent harm to the aerial parts of the host [1]. Foliar tissues of all plant species examined to date harbor at least one and, more commonly, a wide diversity of fungi lurking within their plant tissues [2]. These fungi phylogenetically span at least four phyla [3] and are categorized into two groups based on their manner of transmission: vertically transmitted endophytes (VTEs, e.g., Neotyphodium Glenn et al.) that inhabit temperate grasses (Lolium L.) and are transmitted from the mother plant [4] and horizontally transmitted endophytes (HTEs) transmitted mostly via spores from plant to plant via rain, wind, local leaf litter, or insect vectors [5,6]. HTEs are so common that the foliar tissues of all plant species examined thus far harbor at least one fungus [2]. Although the majority of these endophytic fungi are localized infections by either latent pathogens or dormant saprophytes [2], in many cases endophytes may promote host plant growth, improve nutrient supply and protect plants from both biotic and abiotic stresses [7,8]. Because a plant can be colonized by endophytic fungi that originate from a neighbor or by spores that persist in the local environment, it is not surprising that endophyte communities found in the same plant host species can show striking geographical differences [6].
Non-native (alien, exotic, introduced) species are species whose presence in a region is attributable to human actions that enabled them to overcome fundamentally biogeographic barriers. Only a fraction of non-native species become invasive; they are defined as species that become naturalized, that is, sustain self-replacing populations over several life cycles

Sample Collection and Isolation of Fungi
The Colletotrichum strains used in this study were collected from healthy and diseased leaves of the invasive plant Ageratina adenophora, as well as diseased leaves of native plants, from 2012 to 2018, using leaf fragment incubation [32,48]. Briefly, for the isolation of leaf spot fungi, healthy leaf tissues and the margins of diseased tissues of each leaf spot were cut into 6 mm 2 sections and surface sterilized as follows. These sections underwent initial immersion for 2 min in 0.5% sodium hypochlorite, followed by 1 min in sterile distilled water, 2 min in 75% ethanol, and, finally, 1 min in sterile distilled water. The disinfected fragments were then plated onto potato dextrose agar (PDA; 200 g potato, 20 g glucose, 18 g agar, 1 L distilled water) and incubated at ambient temperature for 6-8 days or until mycelia growing from the leaf fragments were observed. In total, over 1000 Colletotrichum strains were collected and we selected 44 representative strains for further study.
The pure cultures and dried cultures were deposited in the Herbarium of the Laboratory for Conservation and Utilization of Bio-resources, Yunnan University, Kunming, Yunnan, China (YMF) and the China General Microbiological Culture Collection Center (CGMCC).

Morphological Characterization
The growth rates of Colletotrichum isolates were determined on four common media: cornmeal dextrose agar (CMA; 20 g cornmeal, 18 g agar, 1 L distilled water), low nutrient agar (SNA; 1 g KH 2 PO 4 , 1 g KNO 3 , 0.5 g KCl, 0.5 g MgSO 4 , 0.2 g sucrose, 0.2 g glucose, 18 g agar, 1 L distilled water), malt extract agar (MEA; 30 g malt powder, 3 g peptone, 18 g agar, 1 L distilled water), and PDA, respectively. Six-millimeter plugs of actively growing mycelia were inoculated onto these media (20 mL per plate); these cultures were incubated at 25 • C for 7 days. During this time, the colony diameters and characteristics, such as colony appearance, color, and sporogony, on the four media were recorded. Moreover, microscopic characteristics were identified by photographing with a BX51 microscope connected to a DP Controller digital camera. Microscopic characteristics were created from cultures growing on CMA or SNA at 25 • C.
PCR products were firstly checked by performing 1.5% agarose gel electrophoresis stained with ethidium bromide, then purified using the PCR product purification kit (Biocolor BioScience and Technology Co., Shanghai, China), and finally sent for both forward and reverse sequencing on an ABI 3730 XL DNA sequencer (Applied Biosystems, Foster City, CA, USA) with the same primers at Tsingke Biotechnology Co., Ltd. (Kunming, China). Then, the forward and reverse sequences were assembled to obtain a consensus sequence using DNAMAN v. 9.0. These newly generated sequences were deposited in the Gen-Bank database, and the accession numbers from this study in the National Center for Biotechnology Information (NCBI) are listed in Supplementary Table S1.

Multigene Phylogenetic Analyses
The phylogenetic tree was constructed with five loci ACT, CHS-1, GAPDH, TUB2, and ITS to identify these Colletotrichum isolates to the species level. Additional reference species sequences were selected based on recent studies on Colletotrichum species [55,56], referring to 14 species complexes and 13 singleton species. Both the reference sequences and new generated sequences in this study are listed in Supplementary Table S1. Multiple sequence alignments for the five loci were constructed and carried out using Clustalx v. 1.83 [57] with the default settings. The phylogeny was developed based on maximum-likelihood for all individual loci. Then, five datasets of ACT, CHS-1, GAPDH, TUB2, and ITS of Colletotrichum spp. were manually adjusted and linked through BioEdit v. 7.0 [58]. Of these, gap adjustments were performed and ambiguously aligned regions were also excluded. The final concatenated sequence matrix (Fasta file) generated by BioEdit v. 7.0 contained 2119 characters with gaps, ACT with 310 sites, CHS with 299 sites, GAPDH with 363 sites, TUB2 with 532 sites, and ITS with 615 sites. Gaps in the concatenated dataset were treated as missing data in maximum-likelihood (ML) trees and Bayesian inference (BI) methods.
ML analysis was performed using RAxML v. 8.2.10 [59] with the PHY files, which were generated using CLUSTAL_X v. 1.83 [57]. Final ML searches were conducted using the GTR-GAMMA model. Maximum likelihood bootstrap proportions (MLBP) were computed with 1000 replicates and were given above each node of the phylogenetic tree when it was equal to or greater than 70. For BI analysis, a Markov Chain Monte Carlo (MCMC) algorithm was used to generate phylogenetic trees using MrBayes v. 3.1.2 [60]. The concatenated dataset was converted to a NEXUS file with MEGA6 [61], then the Akaike information criterion (AIC) was implemented by using jModelTest v. 2.0 [62] to select the best fit models of nucleotide substitution. GTR+I+G+F was selected as the best fit model for the concatenated dataset. Two independent analyses with four chains were run for 4,000,000 generations sampling every 1000th generation, halting the analyses when the average standard deviation of split frequencies was less than 0.01. The first 25% of the tree generations were discarded as the burn-in phase of the analyses, and the Bayesian inference posterior probability (BIPP) was obtained from the remaining trees. Trees generated in this study were viewed with TreeView v. 1.6.6 [63], and the final topology of phylogenetic trees was based on maximum likelihood analysis, with MLBP greater than 70 and BIPP greater than 0.9, as shown at the nodes.

Multi-Locus Phylogeny
All Colletotrichum spp. isolates were first identified at genus level based on their ITS sequences. To further identify these isolates at species level, we conducted multi-locus phylogenetic analysis referring to 2119 characters and 220 sequences. For the five-locus (CHS-1, GAPDH, ITS, ACT, and TUB2) phylogenetic analysis of the genus Colletotrichum, 173 reference sequences obtained from GenBank and our selected 46 representative sequences of Colletotrichum isolates were used in this study. Monilochaetes infuscans Harter CBS 869.96 was selected as the outgroup according to recent publications [55,56]. When the five individual sequence datasets did not show any conflicts in the tree topologies based on The results of this analysis revealed that 46 isolates were distributed in eight species complex clades, which were, respectively, the C. dracaenophilum species complex, the C. magnum species complex, the C. orchidearum species complex, the C. gigasporum species complex, the C. gloeosporioides species complex, the C. boninense species complex, the C. acutatum species complex, and the C. graminicola-caudatum species complex clades, and one isolate cluster with the singleton species C. sydowii CBS 135819.
For the isolates in the C. dracaenophilum species complex, one isolate clustered with C. excelsum-altitudinum and C. tongrenense (MLBP 81/ BIBP 0.98); we designated the isolate as C. robustum.
For the isolates in the C. magnum species complex, two isolates clustered with the C. magnum clade (MLBP 80) corresponding to the known species C. magnum. For the isolates in the C. orchidearum species complex, two isolates were grouped with the C. plurivorum clade (100/1) and identified as the known species C. plurivorum.
For the isolates in the C. gigasporum species complex, two isolates clustered with the C. gigasporum clade (100/1) corresponding to C. gigasporum.
For the isolates in the C. dracaenophilum species complex, one isolate clustered with C. excelsum-altitudinum and C. tongrenense (MLBP 81/ BIBP 0.98); we designated the isolate as C. robustum.
For the isolates in the C. magnum species complex, two isolates clustered with the C. magnum clade (MLBP 80) corresponding to the known species C. magnum. For the isolates in the C. orchidearum species complex, two isolates were grouped with the C. plurivorum clade (100/1) and identified as the known species C. plurivorum.
For the isolates in the C. gigasporum species complex, two isolates clustered with the C. gigasporum clade (100/1) corresponding to C. gigasporum.
Culture characteristics: Colonies on MEA flat with entire margin, occupied the whole plate in 7 days at 25 • C, surface buff, partly covered with a short, felty, whitish aerial mycelium, reverse medium buff to pale honey. Colonies on PDA flat with entire margin, occupied the whole plate in 7 days at 25 • C, aerial mycelium fluffy, flocculent, white in edges, reverse white to light yellow. Colonies on SNA flat with entire margin, occupied the whole plate in 7 days at 25 • C, covered with thin, whitish aerial mycelium, producing pale gray conidiomata in 2 weeks, reverse same colors. Colonies on CMA reaching 59-60 mm diameter in 7 days at 25 • C, aerial mycelium fluffy, flocculent, white, reverse same colors.
Culture characteristics: Colonies on MEA flat with entire margin, reaching 48-50 mm diameter in 7 days at 25 • C, aerial mycelium fluffy, milky white, reverse same colors. Colonies on PDA reaching 84-88 mm diameter in 7 days at 25 • C, aerial mycelium dense, cottony, gray in the center and white in the margin, reverse grey to saffron with brown pigmentation ring. Colonies on SNA flat with entire margin, reaching 49-52 mm diameter in 7 days at 25 • C, medium buff to pale honey in the center, aerial mycelium fluffy, flocculent, pale yellow, white in edges, reverse buff to pale honey. Colonies on CMA reaching 39-40 mm diameter in 7 days at 25 • C, towards the margin honey to buff, partly covered with short felty whitish aerial mycelium, reverse olivaceous buff to olivaceous gray.
Culture characteristics: Colonies on MEA flat forming a concentric ring with entire margin, occupied the whole plate in 7 days at 25 °C, surface grayish white, aerial mycelia medium hyaline to gray in the center, light gray to grayish white in the margin, partly covered with short felty whitish aerial mycelium, reverse same colors. Colonies on PDA flat forming a concentric ring with entire margin, occupied the whole plate in 7 days at 25 °C, aerial mycelia cotton-shaped, white to pale gray with circular margin, reverse same colors. Colonies on SNA flat with entire margin, occupied the whole plate in 7 days at 25 Sexual morph not observed. Asexual morph on CMA. Conidiomata rudimentary, forming smaller aggregates of the mycelium with conidia droplets, dark olivaceous brown. Setae and conidiophores formed directly on hyphae. Setae scarce, cylindrical, straight or slightly flexuous, dark brown to black, 4-6-septate, base cylindrical to slightly inflated, tip acute to rounded, up to 102 µm. Conidiophores pale brown, aseptate, sometimes branched. Conidiogenous cells hyaline, smooth-walled, septate, subulate or cylindrical. Conidia hyaline, smooth-walled, cylindrical, slightly constricted at the middle, apex rounded, base obtuse, subtruncate, 15.5-20 × 4.5-5.5 µm, mean ± SD = 17.6 ± 1.3 × 5.1 ± 0.3 µm, l/w ratio = 3.5. Appressoria produced from mycelia, irregular, brown, branched, 12-16 × 6-11.5 µm.
Culture characteristics: Colonies on MEA flat forming a concentric ring with entire margin, occupied the whole plate in 7 days at 25 • C, surface grayish white, aerial mycelia medium hyaline to gray in the center, light gray to grayish white in the margin, partly covered with short felty whitish aerial mycelium, reverse same colors. Colonies on PDA flat forming a concentric ring with entire margin, occupied the whole plate in 7 days at 25 • C, aerial mycelia cotton-shaped, white to pale gray with circular margin, reverse same colors. Colonies on SNA flat with entire margin, occupied the whole plate in 7 days at 25 • C, surface covered with a radial, groove, thin, whitish aerial mycelium, reverse same colors. Colonies on CMA reaching 40-43 mm diameter in 7 days at 25 • C, aerial mycelium fluffy, flocculent, white, reverse same colors.
Notes: Based on multi-locus phylogenetic analysis, three strains of Colletotrichum cangyuanense formed a sole clade in the gloeosporioides species complex, and the clade is close to C. psidii Curzi. Morphologically, C. cangyuanense shares similar morphological characteristics with other taxa in the gloeosporioides species complex, which have cylindrical conidia with rounded ends tapering slightly towards the base [66]. Nevertheless, C. cangyuanense is morphologically different from the phylogenetically nearest species C. psidii by having larger conidia, 15.5-20 × 4.5-5.5 vs. 12-15 × 3.5-4.5 µm [66].
Culture characteristics: Colonies on MEA flat with entire margin, reaching 80 mm diameter in 7 days at 25 • C, surface grayish white, partly covered with fluffy, whitish aerial mycelium, light gray to grayish white in the margin, reverse medium grayish white to pale honey with brown pigmentation ring. Colonies on PDA flat with entire margin, occupied the whole plate in 7 days at 25 • C, aerial mycelia cotton-shaped, white to pale gray with circular margin, reverse iron grey to olivaceous grey. Colonies on SNA flat with entire margin, reaching 63-66 mm diameter in 7 days at 25 • C, aerial mycelia medium hyaline to grayish white in center, white to light gray in the margin, partly covered with short felty whitish aerial mycelium, reverse same colors. Colonies on CMA reaching 78 mm diameter in 7 days at 25 • C, aerial mycelium fluffy, flocculent, white, reverse same colors.
Culture characteristics: Colonies on MEA flat with entire margin, occupied the whole plate in 7 days at 25 • C, aerial mycelium fluffy, milky white, reverse brown to buff. Colonies on PDA reaching 72-76 mm diameter in 7 days at 25 • C, aerial mycelium dense, cottony, gray in the center and white in the margin, reverse same colors. Colonies on SNA flat with entire margin, reaching 70-72 mm diameter in 7 days at 25 • C, aerial mycelia medium hyaline to grayish white in center, white in the margin, partly covered with short whitish aerial mycelium, reverse same colors. Colonies growing on CMA with entire margin, reaching 64-66 mm diameter in 7 days at 25 • C, aerial mycelia medium gray to pale buff in center, light gray to grayish white in the margin, entirely covered with floccose to dense, producing pale gray conidiomata in 2 weeks. Reverse dark white to gray with white margin.
Notes: Based on multi-locus phylogenetic analysis, two strains of Colletotrichum gracile formed a solitary clade in the gloeosporioides complex clade, and phylogenetically near to the two new species C. oblongisporum and C. nullisetosum. However, C. gracile is easily distinguished from them by observing sexual morph.
Culture characteristics: Colonies on MEA flat with entire margin, reaching 68-72 mm diameter in 7 days at 25 • C, surface covered with radial, groove, felty, whitish aerial mycelium, Conidiomata acervular, with orange conidial masses, reverse iron gray to buff. Colonies on PDA flat with entire margin, reaching 76-79 mm diameter in 7 days at 25 • C, aerial mycelium dense, cottony, gray in the center and white in the margin, reverse carneose to grayish white. Colonies on SNA flat with entire margin, reaching 70-73 mm diameter in 7 days at 25 • C, aerial mycelia medium hyaline to grayish white in the center, white in the margin, partly covered with short whitish aerial mycelium, producing conidiomata with yellow conidial masses in 2 weeks, reverse same colors. Colonies growing on CMA with entire margin, 73-74 mm diameter in 7 days at 25 • C, aerial mycelia medium gray to pale buff in center, light gray to grayish white in the margin, entirely covered with floccose to dense, producing pale gray conidiomata in 2 weeks. Reverse dark white to grey with white margin.
with entire margin, 73-74 mm diameter in 7 days at 25 °C, aerial mycelia medium gray to pale buff in center, light gray to grayish white in the margin, entirely covered with floccose to dense, producing pale gray conidiomata in 2 weeks. Reverse dark white to grey with white margin.
Additional specimen examined: China, Yunnan province, Nanhua county, from disease spot of Ageratina adenophora, August 2017, Han-Bo Zhang, living culture YMF 1.04990.  Notes: Phylogenetically, Colletotrichum nanhuaense fell into the gloeosporioides complex clade and near to C. gloeosporioides and the new species C. dimorphum. Nevertheless, C. nanhuaense is morphologically different from them by having setae [67].
Colletotrichum nullisetosum Z.F. Yu (Figure 8). Notes: Phylogenetically, Colletotrichum nanhuaense fell into the gloeosporioides complex clade and near to C. gloeosporioides and the new species C. dimorphum. Nevertheless, C. nanhuaense is morphologically different from them by having setae [67].
Culture characteristics: Colonies on MEA reaching 44-45 mm diameter in 7 days at 25 • C, aerial mycelia cotton-shaped, grayish white to white, turning iron gray in the later stage, reverse same colors. Colonies on PDA reaching 49-51 mm diameter in 4 days at 25 • C, aerial mycelia cotton-shaped, white to pale gray with circular margin, reverse iron gray to olivaceous gray. Conidial mass whitish to pale yellow. Colonies on SNA reaching 58-60 mm diameter in 7 days at 25 • C, aerial mycelium fluffy, flocculent, white, producing black conidiomata in 2 weeks, reverse same colors. Colonies on CMA reaching 57-60 mm diameter in 7 days at 25 • C, aerial mycelium fluffy, flocculent, white, reverse same colors.
Additional specimen examined: China, Yunnan province, Ning'er county, from disease spot of the common fruit mango, August 2017, Han-Bo Zhang, living culture YMF 1.07328.
Notes: Colletotrichum nullisetosum is phylogenetically close to the new species C. oblongisporum, but it can be distinguished from C. oblongisporum by having dimorphic conidia.
Colletotrichum oblongisporum Z.F. Yu (Figure 9). Notes: Based on multi-locus phylogenetic analysis, two strains of Colletotrichum oblongisporum formed a solitary clade in the gloeosporioides complex clade, and phylogenetically near to the two new species C. oblongisporum and C. nullisetosum, but it can be easily distinguished from them in morphology. For instance, C. gracile differs from C. oblongisporum in the shape of conidia and appressoria. C. gracile has cylindrical to clavate conidia and subglobose to ellipsoidal appressoria, while C. oblongisporum has oblong co-  Culture characteristics: Colonies on MEA flat with entire margin, occupied the whole plate in 7 days at 25 • C, aerial mycelium fluffy, flocculent, white on the edges, reverse white to light yellow. Colonies on PDA flat with entire margin, occupied the whole plate in 7 days at 25 • C, aerial mycelium dense, cottony, in the later stage of culture, formed a four concentric ring pattern, from the center to the edge, pale gray to dark gray. Colonies on SNA flat with entire margin, occupied the whole plate in 7 days at 25 • C, aerial mycelium white, formed in the shape of four concentric ring pattern, reverse same colors. Colonies on CMA flat with entire margin, occupied the whole plate in 7 days at 25 • C, aerial mycelium fluffy, flocculent, white, reverse same colors.
Notes: Based on multi-locus phylogenetic analysis, two strains of Colletotrichum oblongisporum formed a solitary clade in the gloeosporioides complex clade, and phylogenetically near to the two new species C. oblongisporum and C. nullisetosum, but it can be easily distinguished from them in morphology. For instance, C. gracile differs from C. oblongisporum in the shape of conidia and appressoria. C. gracile has cylindrical to clavate conidia and subglobose to ellipsoidal appressoria, while C. oblongisporum has oblong conidia and subglobose and cuneiform to irregular appressoria.
Culture characteristics: Colonies on MEA reaching 55-58 mm diameter in 7 days at 25 • C, aerial mycelia cotton-shaped, grayish white to white, turning to iron gray in the later stage, reverse same colors. Colonies on PDA reaching 40-49 mm diameter in 5 days at 25 • C, felted aerial mycelia with grayish white appearance, reverse grayish white. Colonies on SNA flat with entire margin, reaching 65-69 mm diameter in 7 days at 25 • C, aerial mycelia medium hyaline to grayish white in center, white in the margin, partly covered with short whitish aerial mycelium, reverse same colors. Colonies on CMA flat with entire margin, reaching 50-54 mm diameter in 7 days at 25 • C, aerial mycelia hyaline to greyish white in center, white in the margin, partly covered with short whitish aerial mycelium, reverse same colors. clavate or irregular outline, aseptate, brown, 7.5-17 × 4.5-10 μm, mean ± SD = 11.7 ± 2.1 × 7.2 ± 1.6 μm. Culture characteristics: Colonies on MEA reaching 55-58 mm diameter in 7 days at 25 °C, aerial mycelia cotton-shaped, grayish white to white, turning to iron gray in the later stage, reverse same colors. Colonies on PDA reaching 40-49 mm diameter in 5 days at 25 °C, felted aerial mycelia with grayish white appearance, reverse grayish white. Colonies on SNA flat with entire margin, reaching 65-69 mm diameter in 7 days at 25 °C, aerial mycelia medium hyaline to grayish white in center, white in the margin, partly covered with short whitish aerial mycelium, reverse same colors. Colonies on CMA flat with entire margin, reaching 50-54 mm diameter in 7 days at 25 °C, aerial mycelia hyaline to greyish white in center, white in the margin, partly covered with short whitish aerial mycelium, reverse same colors. Notes: Phylogenetically, Colletotrichum parvisporum clustered with C. pandanicola Tibpromma and K.D. Hyde in the gloeosporioides complex clade. C. pandanicola was introduced by Tibpromma et al. in 2018; it was recorded as an endophyte isolated from the leaves of Pandanus sp. in Thailand and sexual morph was not reported [68]. Morphologically, C. pandanicola is easily distinguished from the new species C. parvisporum by having cylindrical conidia and lacking appressoria [68].
Notes: Phylogenetically, Colletotrichum parvisporum clustered with C. pandanicola Tibpromma and K.D. Hyde in the gloeosporioides complex clade. C. pandanicola was introduced by Tibpromma et al. in 2018; it was recorded as an endophyte isolated from the leaves of Pandanus sp. in Thailand and sexual morph was not reported [68]. Morphologically, C. pandanicola is easily distinguished from the new species C. parvisporum by having cylindrical conidia and lacking appressoria [68].
Culture characteristics: Colonies on MEA flat forming a concentric ring with entire margin, occupied the whole plate in 7 days at 25 • C, surface grayish white, aerial mycelia medium hyaline to gray in center, light gray to grayish white in the margin, partly covered with short felty whitish aerial mycelium, reverse same colors. Colonies on PDA flat with entire margin, occupied the whole plate in 7 days at 25 • C, aerial mycelium fluffy, flocculent, white in edges, reverse white to light yellow, aerial mycelium sparse, white to pale gray, with copious yellow conidial masses in the later stage of culture, reverse pale luteous to sienna with circinate black colored pigmentation. Colonies on SNA flat is concentric ring with entire margin, occupied the whole plate in 7 days at 25 • C, aerial mycelia medium white to hyaline in center, partly covered with short felty whitish aerial mycelium, reverse same colors. Colonies on CMA flat with entire margin, reaching 68 mm diameter in 7 days at 25 • C, aerial mycelia medium hyaline to grayish white in center, white in the margin, partly covered with a short, whitish aerial mycelium, reverse same colors.
Notes: In this phylogenetic tree, Colletotrichum robustum fell into the dracaenophium complex clade, and clustered with C. excelsum-altitudinum G. Tao, Zuo Y. Liu and L. Cai and C. tongrenense S.X. Zhou, J.C. Kang and K.D. Hyde. However, C. robustum can be distinguished from them in morphology. C. excelsum-altitudinum was isolated as an endophyte from Bletilla ochracea in Guizhou [42]. It has larger conidia and appressoria than C. robustum but has a slower growth rate on PDA. C. tongrenense was also isolated as an endophyte from Nothapodytes pittosporoides in Guizhou [69]. C. tongrenense is obviously different in having shorter conidia, 11-14 × 5-7 µm, and lacking appressoria.
Culture characteristics: Colonies on MEA flat forming a concentric ring with entire margin, occupied the whole plate in 7 days at 25 • C, white to grayish white in the margin, partly covered with short felty whitish aerial mycelium, reverse white to buff. Colonies on PDA reaching 60-63 mm diameter in 7 d at 25 • C, felted aerial mycelia with grayish white appearance, reverse grayish white, aerial mycelia turning dark gray and later becoming cotton-like lately. Colonies on SNA flat with entire margin, reaching 59-61 mm diameter in 7 days at 25 • C, aerial mycelia medium iron gray in center, light gray in the margin, partly covered with short felty gray aerial mycelium, reverse same colors. Colonies on CMA flat with entire margin, reaching 57-61 mm diameter in 7 days at 25 • C, aerial mycelia dark brown, conidiomata scarced, dark, reverse same colors.
Notes: Based on multi-locus phylogenetic analysis, three strains of Colletotrichum simulanticitri formed a solitary clade in the gloeosporioides complex clade and near to C. nymphaeae (Pass.) Aa and C. citri F. Huang, L. Cai, K.D. Hyde and Hong Y. Li. Morphologically, C. simulanticitri can be distinguished from C. nymphaeae by having setae and and shorter conidia (10-13.5 µm vs. 14-18.5 µm) [70]. C. simulanticitri is distinguished from C. citri by the conidia shape and size. The shape conidia of C. citri is fusiform and acute to near-rounded at two ends, while C. simulanticitri is cylindrical to oblong and attenuate at the base and rounded at the apex [38]. Moreover, the conidia of C. simulanticitri are wider than C. citri, 4-5 µm vs. 3.3-4.4 µm.
Culture characteristics: Colonies on MEA flat forming concentric ring with radial groove, reaching 65-67 mm diameter in 7 days at 25 °C, covered with short, felty, whitish aerial mycelium, reverse white to buff. Colonies on PDA reaching 59-61 mm diameter in Culture characteristics: Colonies on MEA flat forming concentric ring with radial groove, reaching 65-67 mm diameter in 7 days at 25 • C, covered with short, felty, whitish aerial mycelium, reverse white to buff. Colonies on PDA reaching 59-61 mm diameter in 7 days at 25 • C, covered with felty aerial mycelium, whitish to pale gray with circular margin, reverse olivaceous gray to pale gray. Colonies on SNA flat with entire margin, reaching 58-60 mm diameter in 7 days at 25 • C, aerial mycelia medium grayish white in the center, light gray to hyaline in the margin, reverse pale gray to hyaline. Colonies on CMA flat with entire margin, reaching 59 mm diameter in 7 days at 25 • C, covered with thin whitish aerial mycelium, producing dark conidiomata in 2 weeks, reverse same colors.
Notes: The phylogenetic analyses based on multi-locus revealed that Colletotrichum speciosum belongs to the acutatum species complex and is closely related to C. chrysanthemi (Hori) Sawada and C. carthami (Fukui) S. Uematsu, Kageyama, Moriwaki & Toy. Sato. However, C. chrysanthemi is morphologically different from C. speciosum by its very short acute-ended conidia [70]. Furthermore, C. carthami also differs from C. speciosum by having fusiform to oblong and narrower (2.6-4.6 µm) conidia [71].
the center, light gray to hyaline in the margin, reverse pale gray to hyaline. Colonies on CMA flat with entire margin, reaching 59 mm diameter in 7 days at 25 °C, covered with thin whitish aerial mycelium, producing dark conidiomata in 2 weeks, reverse same colors. Notes: The phylogenetic analyses based on multi-locus revealed that Colletotrichum speciosum belongs to the acutatum species complex and is closely related to C. chrysanthemi (Hori) Sawada and C. carthami (Fukui) S. Uematsu, Kageyama, Moriwaki & Toy. Sato. However, C. chrysanthemi is morphologically different from C. speciosum by its very short acute-ended conidia [70]. Furthermore, C. carthami also differs from C. speciosum by having fusiform to oblong and narrower (2.6-4.6 μm) conidia [71].
Culture characteristics: Colonies on MEA flat with entire margin, occupied the whole plate in 7 days at 25 • C, aerial mycelium fluffy, milky white, reverse pale gray to buff. Colonies on PDA reaching 59-62 mm diameter in 7 days at 25 • C, aerial mycelium dense, cottony, gray in the center and white in the margin, turning dark later, reverse olivaceous to grayish white. Colonies on SNA flat with entire margin, reaching 85-88 mm diameter in 7 days at 25 • C, aerial mycelia medium hyaline to grayish white in the center, white in the margin, partly covered with short, whitish aerial mycelium, reverse same colors. Colonies on CMA reaching 75-78 mm diameter in 7 days at 25 • C, aerial mycelium fluffy, flocculent, white, turning dark later, reverse same colors.
Notes: Based on multi-locus phylogenetic analysis, two strains of Colletotrichum subhenanense formed a solitary clade in the gloeosporioides complex clade, and near to C. henanense F. Liu and L. Cai, C. yulongense C.L. Hou and X.T. Liu, and C. hederiicola Jayaward. Camporesi and K.D. Hyde. Morphologically, C. subhenanense is most similar to C. yulongense in culture characteristics on PAD and SNA, with cylindrical conidia with obtuse to slightly rounded ends, but C. yulongense is obviously distinguished by having septate setae and narrower conidia (4-5 µm) [72].
Culture characteristics: Colonies on MEA flat with entire margin, reaching 38-42 mm diameter in 7 days at 25 • C, aerial mycelia medium buff to white, hyaline in the margin, partly covered with a short, whitish aerial mycelium, reverse buff to pale honey. Colonies on PDA reaching 80 mm diameter in 7 days at 25 • C, aerial mycelium cottony, pale gray in the center, grayish white at the edge, reverse olivaceous to grayish white. Colonies on SNA flat with entire margin, reaching 68-73 mm diameter in 7 days at 25 • C, aerial mycelia medium hyaline to white in center, white in the margin, partly covered with short whitish aerial mycelium, reverse same colors. Colonies growing on CMA with entire margin, 72-74 mm diameter in 7 days at 25 • C, aerial mycelia medium gray to pale buff in center, light gray to grayish white in the margin, entirely covered with floccose to dense, producing pale gray conidiomata in 2 weeks. Reverse same colors.
Notes: In this phylogenetic tree, Colletotrichum yuanjiangense fell into the gloeosporioides complex clade, and near to the two new species, C. nanhuaense and C. dimorphum, and C. gloeosporioides. Morphologically, C. yuanjiangense is most similar to C. nanhuaense in having septate setae and cylindrical conidia, but it can be distinguished by having subglobose or elliptical appressoria with an irregular edge. medium hyaline to white in center, white in the margin, partly covered with short whitish aerial mycelium, reverse same colors. Colonies growing on CMA with entire margin, 72-74 mm diameter in 7 days at 25 °C, aerial mycelia medium gray to pale buff in center, light gray to grayish white in the margin, entirely covered with floccose to dense, producing pale gray conidiomata in 2 weeks. Reverse same colors. Notes: In this phylogenetic tree, Colletotrichum yuanjiangense fell into the gloeosporioides complex clade, and near to the two new species, C. nanhuaense and C. dimorphum, and C. gloeosporioides. Morphologically, C. yuanjiangense is most similar to C. nanhuaense in having septate setae and cylindrical conidia, but it can be distinguished by having subglobose or elliptical appressoria with an irregular edge.

Discussion
Although plant invasion is common and intensively studied [73,74], fungi associated with the host have received relatively less attention. A few cases have indicated that diverse endophytes were found in the leaves of invasive plants, so these fungi were co-introduced as well as local infection [16]. Regardless of the case, fungus identity is less known. Invasive plants invade a new range and commonly establish novel associations with local microbes [75,76]. Thus, invasive plants are good sources for novel microbial species. In this study, it is surprising that the diversity of Colletotrichum species was so abundant in Ageratina adenophora. We revealed 26 species associated with seven species complexes, including 14 new species C. adenophorae, C. analogum, C. cangyuanense, C. dimorphum, C. gracile, C. nanhuaense, C. nullisetosum, C. oblongisporum, C. parvisporum, C. robustum, C. simulanticitri, C. speciosum, C. subhenanense, C. yunajiangense and 12 known species C. boninense, C. citri, C. cosmi, C. fioriniae, C. fructicola, C. gigasporum, C. godetiae, C. guajavae, C. karstii, C. magnum, C. nymphaeae, C. plurivorum.
Although Colletotrichum is a common endophyte in most plants, its association with specific fungal taxa or its high relative abundance suggests it has a major effect on plant communities, given that Colletotrichum consistently occurs abundantly in the endophyte communities of A. adenophora. We hypothesized a pathogenic role in A. adenophora. The detection of many Colletotrichum spp., including the 14 novel species, in this study indicated the potential of Colletotrichum spread with A. adenophora invasion. The pathogenicity of these Colletotrichum strains (not shown here) indicated the necessity of investigating fungal endophytes in A. adenphora, as well as other invasive plants.
Recent evidence has indicated that the diversity of pathogens on highly abundant introduced hosts has been positively correlated over time with the geographical range of the introduced species and the diversity of invaded habitats [77]. Invasive species Microstegium vimineum (Trin.) A. Camus has been proven to be infected by Bipolaris spp. Shoemaker in the eastern USA [78]. Day et al. [26] isolated fungi from the roots of invasive Vincetoxicum rossicum (Kleopow) Barbar. (Apocynaceae) plants collected across Ontario, Canada, and indicated that some of them can reduce the total biomass of a native species Solidago Canadensis L., which may be important for mediating plant community structure and the persistence of invasive plants. Several studies have also found accompanying changes in populations of specific soil pathogens and their impacts on invasive and noninvasive species [79,80]. Recently, fungi isolated from the invasive plant Vincetoxicum rossicum have been proven to have differential growth effects between non-native invasive and native plants, which may be important for mediating plant community structure and the persistence of invasive plants [26]. Accumulations of pathogens are common in several detected invasive plants, including above-ground, for example, leaves [78], and belowground, e.g., soil oomycete pathogens in freshwater wetland soils invaded by non-native Phragmites australis (Cav.) Trin. ex Steud. (European common reed) [80]. We found that rhizosphere soils of Chromolaena odorata L., one of the world's most destructive tropical invasive weeds, accumulate high concentrations of the generalist soil-borne fungi, Fusarium Link (tentatively identified as F. semitectum), thus creating a negative feedback for native plant species [79]. If Colletotrichum is pathogenic on A. adenophora, an abundance of this species may alleviate the spread of A. adenophora. If it is pathogenic on native plants, including fruits, the risk is great and should be emphasized in future.
Previously, association with the fungal endophyte Neotyphodium coenophialum (Morgan-Jones and W. Gams) Glenn et al. increased the ability of Lolium arundinaceum (Schreb.) S.J. Darbyshire to invade communities with greater species diversity. In the absence of the endophyte, the initial diversity of the community significantly reduced the establishment of L. arundinaceum. However, establishment was independent of initial diversity in the presence of the endophyte [81]. We tested the impact of the fungal endophyte Alternaria alternata (Fr.) Keissl. (phylotype CID 120) on the allelopathic effect of the invasive forb Centaurea stoebe when in competition with the North American native bunchgrass Koeleria macrantha (Ledeb.) Schultes in a greenhouse competition experiment. The allelopathic effect on K. macrantha of C. stoebe when infected with the fungal endophyte was more than twice that of endophyte-free C. stoebe. Fungal endophytes may increase the competitive ability of their hosts [23]. Two phylotypes of Alternaria endophytes influenced the growth, competitive effects, and competitive responses of the exotic invasive forb C. stoebe. The competitive effects of C. stoebe on grass species native to North America were enhanced by both fungal endophytes [24]. Arnold et al. [40] indicated that Colletotrichum-enhanced cacao trees resist pathogen attack. In this case, the role of the foliar fungus Colletotrichum sp. may be a weak latent foliar pathogen of A. adenophora and can enhance the pathogenicity of the fungus Diaporthe helianthi on the leaves of A. adenophora [31]. However, Colletotrichum may also as an endophyte of A. adenophora to resist herbivores, for example, Fang K [31] inoculated Collectotrichum into A. adenophora to enhance resistance herbivores. Therefore, the role of Colletotrichum in A. adenophora invasion or its interaction with fungal biocontrol agents is very complex and needs more investigation.
In conclusion, our study reported the diversity of Colletotrichum in the leaves of A. adenophora and several surrounding crops in the introduced range in China. There were several significant agronomic findings. Firstly, there is a potential application for controlling weeds in agriculture, although it remains to be clarified whether these endophytic Colletotrichum species interact with chemical herbicides. The fact that Colletotrichum sp. can enhance the pathogenicity of the fungus D. helianthi on the leaves of A. adenophora [31] provides a potential application for biocontrol of A. adenophora by combination of Colletotrichum with pathogenic fungi, such as D. helianthi. Secondly, because the Colletotrichum genus is one of the most important pathogenic fungi [33], our finding highlighted the high disease risk driven by invasive plants in the invaded ecosystem. Indeed, some of these Colletotrichum species were previously verified to be pathogenic on local native plants [32], as well as on tested fruits, including grape and mango, as the recent research by Zheng et al. [82] also revealed. This result suggested that invasive species can facilitate pathogen emergence and amplification, raising concerns about the movement of pathogens among agricultural, horticultural and wild grasses. For example, in all, 39 C. gloeosporioides and 3 C. acutatum isolates were recovered from diseased strawberry crowns, and 52 C. gloeosporioides and one C. acutatum isolate were recovered from non-cultivated hosts. The results were not inconsistent with the hypothesis that C. gloeosporioides isolates obtained from strawberry and noncultivated hosts adjacent to strawberry fields are from the same population and that noncultivated hosts can serve as potential inoculum sources for Colletotrichum crown rot of strawberry [83]. Finally, 14 novel Colletotrichum species were reported in invasive plants A. adenophora and several surrounding crops in south-western China. This result suggested that exotic plants are sources for novel fungal species, and the fungi associated with invasive plants are worth investigating in the future.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/jof8020185/s1. Table S1: Previously described Colletotrichum species and New species used in phylogenetic analyses.  Data Availability Statement: All sequence data are available in NCBI GenBank following the accession numbers in the manuscript.