Identification and Pathogenicity of Pestalotioid Species on Alpinia oxyphylla in Hainan Province, China

Alpinia oxyphylla is a traditional Chinese medicinal plant with a medicinal history of more than 1700 years. Ring leaf blight (RLB) disease, caused by pestalotioid species, is an important disease of A. oxyphylla, seriously affecting the yield and quality of its fruits. The causal agent of RLB disease has not been systematically identified or characterized yet. In this study, thirty-six pestalotioid strains were isolated from the leaves and stems of A. oxyphylla that was collected from six cities of Hainan province, China. Based on the multi-locus phylogeny (ITS, tef-1α and tub2) and morphological characteristic analyses, seventeen species belonging to three genera (Neopestalotiopsis, Pestalotiopsis and Pseudopestalotiopsis) were identified, and six new species (N. baotingensis, N. oblatespora, N. olivaceous, N. oxyphylla, N. wuzhishanensis and N. yongxunensis) were described. Pathogenicity tests revealed that strains of Neopestalotiopsis species caused more severe ring leaf blight on A. oxyphylla than strains of Pestalotiopsis and Pseudopestalotiopsis under wounded inoculation conditions.


Introduction
Alpinia oxyphylla belongs to the family Zingiberaceae and is an important Chinese herbal plant, with a medicinal history dating back 1700 years [1].As an edible herb, the traditional medicinal effects of A. oxyphylla's fruit mainly include warming the kidney, stopping spermatorrhea, arresting polyuria, warming the spleen as well as stopping diarrhea and excess saliva [2,3].Moreover, the essential oil of A. oxyphylla has various effects including antibacterial, anticancer, antioxidant, vasodilation and improved immunity [4]. A. oxyphylla likes to grow in warm and humid environmental conditions and is commonly planted under rubber trees, areca trees and other economic forests as a semi-shade plant [5][6][7].A. oxyphylla is mainly distributed in southern China, such as in Hainan, Guangdong and Guangxi provinces.Among them, Hainan, with abundant rainfall and high temperatures, is the most important planting area for A. oxyphylla, accounting for 90% of the total output in China [8][9][10].
The occurrence of diseases causes serious losses to the production and quality of A. oxyphylla.Ring leaf blight (RLB) is an important disease of A. oxyphylla that occurs from the seedling to the fruiting stage, mainly infecting old leaves.The disease often extends from the leaf edge or tip, forming irregular, reddish-brown spots with alternating dark and light brown wavy concentric rings and obvious yellow halos around the periphery of the disease spots, on which numerous small black conidiomata of the pathogen are scattered.The pathogen of this disease can be transmitted through wind and rain, mainly invading through wounds.The high temperature and rainy season contribute to the occurrence of RLB disease, and the high incidence of this disease is from August to September.Under suitable conditions, the proportion of diseased plants can reach more than 50%, and the area of the diseased spots can reach 1/3-1/2 of the leaf surface, even the entire leaf, which has an impressive impact on the growth of A. oxyphylla [11,12].
The pathogen of RLB disease was first reported as Pestalotia palmarum in 1986 [11].Subsequently, the classification status of P. palmarum was adjusted to the genus Pestalotiopsis, while Pestalotia and Pestalotiopsis were used confusingly in descriptions of A. oxyphylla diseases [13].The ring brown spot (RBS) disease of A. oxyphylla was caused by Pestalosphaeria alpinia, a sexual morph of pestalotioid fungi [14].As asexual fungi, most pestalotioid species lack the sexual morphs Pestalosphaeria [15].Most of pestalotioid species are important plant pathogens and are also commonly found as endophytes or saprophytes, being mainly distributed throughout tropical and temperate regions [16][17][18].Pestalotioid species can infect the leaves, shoots, flowers, fruits or other parts of plants and cause a variety of diseases in multiple economic crops, including leaf spots, gray blight, shoot dieback, trunk diseases, dry flowers and fruit rot [17,[19][20][21][22][23][24][25][26].Hence, pestalotioid species causing disease in A. oxyphylla need to be reidentified and characterized based on their fungal diversity, molecular systematics and pathogenicity.
The development of a molecular phylogenetic analysis overcomes the limitation of overlapping conidial measurements in the traditional taxonomy of pestalotioid species [16,17,27,28].In 2014, two novel genera, Neopestalotiopsis and Pseudopestalotiopsis, were segregated from Pestalotiopsis based on conidial characters and multi-locus phylogenetic analyses.The combined sequences of the ITS, tub2 and tef-1α genes were used to construct phylogenetic trees, which become an important basis for distinguishing different species within the genera Pestalotiopsis, Neopestalotiopsis and Pseudopestalotiopsis. Morphologically, Neopestalotiopsis can be easily differentiated from Pestalotiopsis and Pseudopestalotiopsis by the versicolorous median cells of the conidia, and Pseudopestalotiopsis is different from Pestalotiopsis with its three darker, concolorous median cells [17].Through these methods, many novel pestalotioid species isolated from different plants have been introduced in recent years [19,22,[29][30][31][32][33][34].
Therefore, the objective of this study is to clarify the types, characteristics and pathogenicities of pestalotioid species related to disease in A. oxyphylla of Hainan, China.

Sample Collection, Fungi Isolation and Morphological Examination
Fresh leaves of A. oxyphylla with typical ring spots and stems with irregular cloud-like spots were collected from the main planted areas at ten townships in six cities of Hainan province, including Baoting, Ledong, Qiongzhong, Sanya, Wanning and Wuzhishan in 2022.Small pieces (5 mm × 5 mm) of leaves or stems were cut from the junctions of diseased and healthy areas, disinfected with 3% sodium hypochlorite for 3 min, then 75% ethanol for 30 s and subsequently washed with sterilized water three times.The treated tissue pieces were dried on sterilized blotting paper and then placed on PDA plates (containing 100 µg/mL streptomycin, 50 µg/mL kanamycin and 100 µg/mL ampicillin).The plates were cultured at room temperature and examined daily for 7 days; then, the marginal mycelia with different morphologies on each plate were transferred to fresh PDA; subsequently, the pestalotioid strains were purified using single-spore culturing according to the results of the ITS sequence analysis.
The pestalotioid strains usually sporulated at room temperature on PDA after 10-20 days.The conidiomata were observed using a dissecting microscope (CNOPTEC, SZ680, Chongqing, China), and the characteristics of spores and conidiophores were observed using an optical microscope (CNOPTEC, DV320, Chongqing, China).All the morphological characteristics of the spores were photographed and measured for at least 30 individuals using OPTPro     Ex-type strains are labeled with T .NA: not available.The strains in this study are indicated in bold font.
The phylogenetic analyses of the combined sequences were carried out using maximumlikelihood (ML) and Bayesian inference (BI) methods.The ML analysis was performed on the CIPRES web portal (https://www.phylo.org,accessed on 31 October 2023) using RAxML-HPC BlackBox 8.2.10, with a GTRGAMMA substitution model and 1000 bootstrap replicates [91].The BI analysis was implemented using MrBayes v.3.2.7 [92], and MrModeltest 2.2 [92] was used to seek the best-fit nucleotide substitution models for each gene.Two Markov chain Monte Carlo (MCMC) methods were run for 1,000,000 generations, and trees were sampled every 1000th generation.The first 25% of trees, standing for the burn-in phase of the analyses, were discarded, and the remaining trees were estimated to be the posterior probabilities.The ML tree and BI tree were viewed using Figtree v.1.4.4.and modified using WPS Office v.12.1.0.16729.
The new species can be further confirmed through PHI (Pairwise Homoplasy Index) analysis, which can also be used to analyze the species' boundaries and related taxa [93].The PHI test was completed using SplitsTree v.4 [94,95], and a value over 0.05 revealed no significant recombination in the dataset.The relationships among closely related species were shown using splits graphs through the LogDet transformation and split decomposition.

Pathogenicity Test
The pathogenicity of the fungi was tested using the wound inoculation method.Fresh and healthy leaves of A. oxyphylla measuring 30-40 cm long were collected from the field.The surface of the leaves was disinfected by spraying them with 75% ethanol and then washed three times with sterile water.Each fungal isolate was inoculated on 6 sites per leaf with 3 leaf replicates.A piece of mycelium (6 mm diameter), which was taken from the margin of a fresh colony cultured to 2/3 of the PDA plate's diameter, was placed on the wound of injured leaf using a sterilized needle.A piece of PDA without mycelium was used as the control.The inoculated leaves were placed in a box and cultured in the incubator at 26 • C and 600 LUX, with a 16 h/8 h LED light/dark cycle.After 5 days, disease symptoms were recorded, the lesion area was measured using ImageJ v.1.53cand the data were analyzed using SPSS Statistics 24.The re-isolated fungi from the disease lesion were identified and tested using Koch's postulates.

Phylogenetic Analyses
A total of 36 pestalotioid isolates were obtained from the leaves (32 isolates) and stems (4 isolates) of A. oxyphylla from six cities in Hainan province.Based on the ITS sequence and color of the intermediate cells of the conidia, 36 strains were classified into three genera, of which 32 strains belong to Neopestalotiopsis, 2 strains belong to Pestalotiopsis and 2 strains belong to Pseudopestalotiopsis.
The phylogenetic tree of Neopestalotiopsis contained 145 taxa, with 2 outgroup taxa (Pestalotiopsis colombiensis and P. diversiseta).A total of 1404 characters, including gaps (503 for ITS, 469 for tef-1a and 432 for tub2), were included in the phylogenetic analysis.For the Bayesian inference, the HKY + G model with a gamma-distributed rate was selected for ITS, the HKY + G model with a gamma-distributed rate was selected for tef1-a and the HKY + I + G model with an invgamma-distributed rate was selected for tub2.Similar tree topologies were acquired using the ML and BI methods, and the best scoring ML tree is shown in Figure 1.The phylogenetic tree depicts 32 Neopestalotiopsis taxa isolated from A. oxyphylla, revealing 6 novel species.
The phylogenetic tree of Pestalotiopsis comprised 78 taxa, with the outgroup taxon N. cubana CBS 600.96.A total of 1457 characters, including gaps (505 for ITS, 495 for tef-1a and 457 for tub2), were included in the phylogenetic analysis.For the Bayesian inference, the GTR + I + G model with an invgamma-distributed rate was selected for ITS, the GTR + G model with a gamma-distributed rate was selected for tef1-a and the GTR + I + G model with an invgamma-distributed rate was selected for tub2.Similar tree topologies were obtained using the ML and BI methods, and the best scoring ML tree is shown in Figure 2. The phylogenetic tree depicts two Pestalotiopsis strains isolated from A. oxyphylla, clustered with the type species of P. hydei.
The alignment of Pseudopestalotiopsis contained 35 taxa, with P. trachicarpicola OP068 as the outgroup taxon.A total of 1392 characters, including gaps (521 for ITS, 442 for tef-1a and 429 for tub2), were included in the phylogenetic analysis.For the Bayesian inference, the HKY + G model with a gamma-distributed rate was selected for ITS, the HKY + G model with a gamma-distributed rate was selected for tef1-a and the HKY + I model with a propinv-distributed rate was selected for tub2.Similar tree topologies were obtained using the ML and BI methods, and the best scoring ML tree is shown in Figure 3.The phylogenetic tree depicts two Pseudopestalotiopsis taxa isolated from A. oxyphylla, clustered with the type species of Ps. avicenniae and Ps.myanmarina, respectively.

PHI Analyses
The results of the PHI test indicate no obvious recombination (Φw = 0.1064) among N. baotingensis SX41-0706, N. oblatespora YJ11-0708 and their closely related species N. saprophytica MFLUCC 12-0282, N. paeoniea CBS 318.74, N. hydeana MFLUCC 20-0132, N. egyptiaca CBS 140162, N. guajavicola FMBCC 11.4 and N. mesopotamica CBS 299.74 (Figure 4a).And there is no significant recombination (Φw = 0.0786) between N. olivaceous LF25-0709 and its closely related species N. amomi HKAS 124563, N. zingiberis GUCC 21001 and N. magna MFLUCC 12-0652 (Figure 4b).N. yongxunensis YX101-0708, N. wuzhishanensis YX116-0708 and their closely taxa have no significant recombination according to the PHI test results (Φw = 0.1103) (Figure 4c).and 457 for tub2), were included in the phylogenetic analysis.For the Bayesian inference, the GTR + I + G model with an invgamma-distributed rate was selected for ITS, the GTR + G model with a gamma-distributed rate was selected for tef1-a and the GTR + I + G model with an invgamma-distributed rate was selected for tub2.Similar tree topologies were obtained using the ML and BI methods, and the best scoring ML tree is shown in Figure 2. The phylogenetic tree depicts two Pestalotiopsis strains isolated from A. oxyphylla, clustered with the type species of P. hydei.The alignment of Pseudopestalotiopsis contained 35 taxa, with P. trachicarpicola OP068 as the outgroup taxon.A total of 1392 characters, including gaps (521 for ITS, 442 for tef-1a and 429 for tub2), were included in the phylogenetic analysis.For the Bayesian inference, the HKY + G model with a gamma-distributed rate was selected for ITS, the HKY + G model with a gamma-distributed rate was selected for tef1-a and the HKY + I model with a propinv-distributed rate was selected for tub2.Similar tree topologies were

Taxonomy
Based on the multi-locus phylogeny (ITS, tef-1α and tub2) and morphological characteristic analyses, 17 species were identified.Three Neopestalotiopsis strains failed to acquire spores and were not identified as specific species.Six new species are described below.The conidial dimensions of the identified isolates in this study and their closely related strains are shown in Table 3.
Neopestalotiopsis baotingensis X.F.Cui and Z.G.Hao, sp.nov.(Figure 5).MycoBank: MB854050.Etymology: It is named in reference to the first collection city of Baoting in Hainan Province.
Culture characteristics: The colony reached 70 mm in diameter on PDA after 4 days of growth at room temperature.The colony was off white, with dense aerial hyphae on the surface with crenate edges, and its reverse was lemon yellow.
Material examined: The sample originated in China, Hainan Province, Baoting city, Shiling Township, Shuixian village, from leaf spots of A. oxyphylla, which was collected

Taxonomy
Based on the multi-locus phylogeny (ITS, tef-1α and tub2) and morphological characteristic analyses, 17 species were identified.Three Neopestalotiopsis strains failed to acquire spores and were not identified as specific species.Six new species are described below.The conidial dimensions of the identified isolates in this study and their closely related strains are shown in Table 3.
Neopestalotiopsis baotingensis X.F.Cui and Z.G.Hao, sp.nov.(Figure 5).MycoBank: MB854050.Etymology: It is named in reference to the first collection city of Baoting in Hainan Province.

Description:
The conidiomata on PDA are solitary or aggregated, globose and dark.The conidiophores often degenerated to conidiogenous cells.Conidiogenous cells are spherical and hyaline.The conidia are fusiform, straight to slightly curved, 18-26 × 5-7.2 µm (x = 23.2 × 6.3 µm) and have four septa.The basal cell is conical to obtuse, hyaline, thin and smooth walled and is 3.2-6.2µm long (x = 4.5 µm).The three median cells are 12-17.3µm (x = 14.8 µm), verruculose, versicolored, pale brown to dark brown and the septa and periclinal walls are darker than the rest of the cell.The second cell from the base is pale brown to brown, is paler than the two other cells and is 3.2-5.5 µm long (x = 4.4 µm).The third cell is brown to dark brown, darker than the two other cells and is 4-6 µm long (x = 4.9 µm).The fourth cell is brown to dark brown and 4-6 µm long (x = 5 µm).The apical cell is 2.5-5 µm long (x = 3.8 µm) and cylindric to subcylindric, with 2-4 tubular appendages on it, often 2-3, arising from its apex, which are unbranched and 3-30.5 µm long (x = 19.7 µm).The single basal appendage is unbranched, tubular, centric and 2.5-10 µm long (x = 6.3 µm).A sexual morph was not observed.
Culture characteristics: The colony reached 70 mm in diameter on PDA after 4 days of growth at room temperature.The colony was off white, with dense aerial hyphae on the surface with crenate edges, and its reverse was lemon yellow.
Culture characteristics: The colonies reached 70 mm in diameter after 4 days on PDA at room temperature, and had serrated-edge, off-white, sparse aerial hyphae on the surface appearing to radiate, turning grey after sporulation.
Culture characteristics: The colonies reached 70 mm in diameter on PDA after 7 days of growth at room temperature.The colonies appeared circular, white above and medium Neopestalotiopsis olivaceous X.F.Cui and Z.G.Hao, sp.nov.(Figure 7).MycoBank: MB854052.Etymology: The name refers to the color of the colony.Holotype: LF25-0709.Description: Conidiomata were not observed on PDA.The conidia sometimes aggregate, becoming globose, dark green piles.The conidiophore are branched, with spore scars.The conidia are fusiform, straight to obviously irregularly curved, 21.5-33.8× 5.5-7.7 µm (x = 26.5 × 6.3 µm) and have four septa.The basal cell is conical, hyaline or pale olive, smooth, thin walled and 2.7-6.2µm long (x = 4.5 µm).The three median cells are 14 to 21.7 µm long (x = 17 µm), pale olivaceous to olivaceous, concolorous and have a rugose wall, with septa darker than the rest of the cell.The second cell from the base is pale olivaceous to olivaceous and 3.3 to 8.5 µm long (x = 5.9 µm).The third cell is pale olivaceous to olivaceous and 4 to 6.5 µm long (x = 5.1 µm).The fourth cell is pale olivaceous to olivaceous and 4 to 6.5 µm long (x = 5.4 µm).The apical cell is 3.5 to 5.5 µm long (x = 4.5 µm), hyaline, and conic to acute, with 2 to 5 (often 3-4) tubular appendages on the apical cell, which are inserted at different loci in a crest at the apex of the apical cell, unbranched and 9.5 to 22.5 µm (x = 14 µm) long.A single basal appendage, which is occasionally absent, is unbranched, tubular, centric or lateral and 1.2 to 4.8 µm (x = 2.4 µm) long.A sexual morph was not observed.
Culture characteristics: The colonies reached 70 mm in diameter on PDA after 7 days of growth at room temperature.The colonies appeared circular, white above and medium dense, with aerial hyphae on the flat surface; its reverse was olivaceous, gradually deepening over time.Notes: Three strains of Neopestalotiopsis olivaceous were isolated from three cities in Hainan, LF25-0709, SX33-0706 and YX45-0708, with well-supported clusters (ML = 99%,   The third cell is pale brown and 3.5-5.2µm long (x = 4.4 µm); the fourth is pale brown and 3.8-6.3µm long (x = 4.7 µm).The apical cell is 2.7-5.5 µm long (x = 3.6 µm), conic to acute, hyaline, thin and smooth-walled, with 1-3 tubular appendages on the apical cell (often 1-2) arising from the apex of the apical cell, which are unbranched, straight to flexuous and 9-20.8 µm long (x = 15.4 µm).There is a single or no basal appendage, which is unbranched, tubular, centric and 0.8-3.8µm long (x = 1.9 µm).A sexual morph is not observed.
Culture characteristics: The colonies reached 70 mm in diameter after 12 days on PDA at room temperature, with circular-edge, white, medium-dense, aerial hyphae on the flat surface, with a filiform margin, black and fruiting bodies.And its reverse was lemon yellow.
Material examined: The sample originated in China, Hainan Province, Wuzhishan city, Shuiman Township, Yongxun village, from leaf spots of A. oxyphylla, which was collected on 8 July 2022 by X.F.Cui and Z.G.Hao (YX116-0708).

Etymology:
The name refers to the first collection village of Yongxun in Hainan Province.

Description:
The conidiomata on the PDA are solitary or aggregated, globose, dark and embedded or semi-immersed.The conidiophores often degenerated to conidiogenous cells.Conidiogenous cells are unclear.The conidia are fusiform, straight to curved, 18.2-25.5× 5.8-7.5 µm (x = 21.6 × 6.6 µm) and have four septa.The basal cell is conical, hyaline, thin and smooth walled and is 3.0-5.2µm long (x = 4.1 µm).The three median cells are 12-15.2µm (x = 13.7 µm), versicolored, pale brown to brown and have septa and periclinal walls that are darker than the rest of the cell; the second cell from the base is pale brown, paler than the other two cells and is 3.5-5.3µm long (x = 4.3 µm).The third cell is brown, darker than the other two and is 3.8-5.3µm long (x = 4.6 µm).The fourth cell is brown and 4.0-5.2µm long (x = 4.6 µm).The apical cell is 2.5-5.0 µm long (x = 3.7 µm), conic to subcylindrical, hyaline, thin and smooth walled, with 2-4 tubular appendages on the apical cell arising from the apex of the apical cell, which are filiform, unbranched, straight to flexuous and 10.5-24.7 µm long (x = 18.2 µm).The single basal appendage is unbranched, tubular, centric and 1.7-7 µm long (x = 4.2 µm).A sexual morph is not observed.
Culture characteristics: The colonies reached 70 mm in diameter after 4 days on PDA at room temperature, with a circular-edge, white, dense aerial mycelium on the surface; the reverse was similar in color.The fruiting bodies were black, mostly under the hyphae and were visible on the back.

Pathogenicity Assay
Sixteen of the twenty tested pestalotioid isolates were able to cause typical brown lesions after inoculation, while the other four isolates did not, including Neopestalotiopsis sp.11).The morphology of the purified fungi re-isolated from the lesion after inoculation was identical with those of the isolates used for inoculation, which were also confirmed using PCR and gene sequences.The results of the pathogenicity and phylogenetic analysis showed that the strains close to N. cubana and N. brachiata had a stronger pathogenicity (Figures 1 and 11B).
on ITS, tef1-α and tub2 sequences, showing a 7 bp difference (2/284 in tef1-α and 5/416 in tub2) with N. dendrobii and 10 bp difference (9/440 in tef1-α and 1/715 in tub2) with N. paeonia-suffruticosa.In addition, there are remarkable discrepancies in the morphological characteristics: N. yongxunensis is thinner in its conidia (N.yongxunensis: 5.8-7.5 µm, ̅ = 6.6 µm vs. N. paeonia-suffruticosa: 9-11 µm, ̅ = 9.5 µm), has different numbers of apical appendages (N.yongxunensis 2-4 vs. N. paeonia-suffruticosa 3-4) and shorter apical appendages (N.yongxunensis: 10.5-24.7 µm vs. N. paeonia-suffruticosa 22.5-34 µm), while N. yongxunensis differs from N. dendrobii in having longer apical appendages (N.yongxunensis: 10.5-24.7 µm vs. N. dendrobii 5-6.5 µm) with different numbers (N.yongxunensis 2-4 vs. N. dendrobii 2-3).Furthermore, the PHI test indicated that there is no significant recombination between N. yongxunensis and its closely related species.Therefore, N. yongxunensis is classified as a new species in the present study.11).The morphology of the purified fungi re-isolated from the lesion after inoculation was identical with those of the isolates used for inoculation, which were also confirmed using PCR and gene sequences.The results of the pathogenicity and phylogenetic analysis showed that the strains close to N. cubana and N. brachiata had a stronger pathogenicity (Figures 1 and 11B).classified as Pestalotiopsis palmarum, while the RBS disease, with similar symptoms to RLB disease, was caused by Pestalosphaeria alpinia, the sexual morph of pestalotioid, as reported in 1994 [15].Perhaps due to the differences in the classification method and limitations in the sample size, P. palmarum and P. alpinia were not isolated in this study, which explained the potential diversity of pestalotioid fungi in this host that need to be further explored.In addition, the symptoms of RLB and RBS disease are similar, with both caused by pestalotioid fungi with different morphs, so it is recommended to merge the two diseases into one for future research and disease management.
The pathogenicity tests of 20 pestalotioid strains showed that most species can cause obvious symptoms on the leaves, indicating the diversity of the pathogen of RLB disease, and the Neopestalotiopsis species (lesion area over 75 mm 2 for 7 species) tended to infect A. oxyphylla and caused more serious disease than Pestalotiopsis (lesion area of about 50 mm 2 ) and Pseudopestalotiopsis (less than 50 mm 2 for both).The reports of the disease caused by Neopestalotiopsis fungi have been more frequent in recent years [18].In addition, all pathogenicity tests were carried out with a single cultivar of A. oxyphylla and constant environmental conditions.As we all know, both differences in varieties and changes in the environmental conditions can affect the occurrence of diseases.Therefore, more studies need to be performed on different varieties under different environmental conditions.
What is worth noting is that most of the pestalotioid species have a broad range of hosts, and one species of pestalotioid fungi can infect several economic plants, while a plant can be harmed by several pestalotioid fungi.For example, N. cubana can infect rubber trees [96], Camellia oleifera [19] and Ixora chinensis [97], and a new leaf fall disease of rubber trees was caused by N. aotearoa, N. cubana and N. formicarum [96]. A. oxyphylla is a semi-shade plant mainly planted in rubber tree forests.In this study, six strains of N. cubana, one strain of Neopestalotiopsis sp.3 SX11-0706 clustered with N. formicarum and five strains (N.oxyphylla, N. brachiata and Neopestalotiopsis sp.5 XC11-0709) closely related to N. aotearoa were isolated.Thus, it suggests that some pestalotioid species may infect both the rubber tree and A. oxyphylla.The promotion of medicinal plant cultivation under forest trees should be carried out with attention to the occurrence of cross-infection diseases in order to prevent them.
A comprehensive understanding of the species and genetic diversity of pathogens is the foundation for sustainable disease management.Since there is no research about the resistance varieties of A. oxyphylla to RLB disease, the strains with different characteristics and pathogenicities that were isolated in this study may provide a material basis for the subsequent screening of resistant varieties, including highly active biological and chemical agents friendly to the environment.

Figure 1 .
Figure 1.RAxML tree of Neopestalotiopsis isolates based on ITS, tef-1α and tub2 sequences.The roots of this tree are Pestalotiopsis diversiseta MFLUCC 12-0287 and P. colombiensis CBS 118553.The strains isolated in this study are marked in red.Ex-type strains are marked with T .ML bootstrap values ≥ 50% and BI probabilities (in red) ≥ 0.90 are displayed at the nodes.The phylogenetic tree of Pestalotiopsis comprised 78 taxa, with the outgroup taxon N. cubana CBS 600.96.A total of 1457 characters, including gaps (505 for ITS, 495 for tef-1a

Figure 1 .
Figure 1.RAxML tree of Neopestalotiopsis isolates based on ITS, tef-1α and tub2 sequences.The roots of this tree are Pestalotiopsis diversiseta MFLUCC 12-0287 and P. colombiensis CBS 118553.The strains isolated in this study are marked in red.Ex-type strains are marked with T .ML bootstrap values ≥ 50% and BI probabilities (in red) ≥ 0.90 are displayed at the nodes.

Figure 2 .
Figure 2. RAxML tree of Pestalotiopsis isolates based on ITS, tef-1α and tub2 sequences.The root of this tree is N. cubana CBS 600.9.The strains isolated in this study are marked in red.Ex-type strains are marked with T .ML bootstrap values ≥ 50% and BI probabilities (in red) ≥ 0.90 are displayed at the nodes.

Figure 2 .
Figure 2. RAxML tree of Pestalotiopsis isolates based on ITS, tef-1α and tub2 sequences.The root of this tree is N. cubana CBS 600.9.The strains isolated in this study are marked in red.Ex-type strains are marked with T .ML bootstrap values ≥ 50% and BI probabilities (in red) ≥ 0.90 are displayed at the nodes.J. Fungi 2024, 10, x FOR PEER REVIEW 14 of 30

Figure 4 .
Figure 4. Split graphs showing the results of the PHI test of new (a) N. baotingensis SX41-0706, N. oblatespora YJ11-0708, (b) N. olivaceous LF25-0709, and (c) N. yongxunensis YX101-0708 and N. wuzhishanensis YX116-0708 with their most closely related species.The new species in each graph is shown in red font.Ex-type strains are marked with " T ".

Figure 4 .
Figure 4. Split graphs showing the results of the PHI test of new (a) N. baotingensis SX41-0706, N. oblatespora YJ11-0708, (b) N. olivaceous LF25-0709, and (c) N. yongxunensis YX101-0708 and N. wuzhishanensis YX116-0708 with their most closely related species.The new species in each graph is shown in red font.Ex-type strains are marked with " T ".

Table 2 .
The strain information and gene accession numbers for pestalotioid species used in this study.

Table 3 .
The conidial dimensions of pestalotioid species related to this study.
The strains in this study are indicated in bold font.NA: not available.