Identification and Pathogenicity of Pestalotiod Fungi Associated with Woody Oil Plants in Sichuan Province, China

Pestalotiod fungi are associated with a wide variety of plants worldwide and occur as endophytes, pathogens, and saprobes. The present study provides an updated phylogeny for genera Neopestalotiopsis, Pestalotiopsis, and Seiridium using fresh collections from woody oil plants (Camellia oleifera, Olea europaea, Paeonia suffruticosa, Sapium sebiferum, and Vernicia fordii) in Sichuan Province, China. We coupled morphology and combined sequence data analyses of ITS, tub2, and tef1-α for Neopestalotiopsis and Pestalotiopsis, with ITS, LSU, tub2, tef1-α, and rpb2 for Seiridium. Three novel species of Neopestalotiopsis (N. mianyangensis, N. paeonia-suffruticosa, N. terricola) and three of Seiridium (S. guangyuanum, S. vernicola, S. oleae), were found. Three other species, Pestalotiopsis kenyana, Seiridium ceratosporum, and S. rosarum were identified and reported as new records. All isolated species are fully described and illustrated. Additionally, the sexual morph of Pestalotiopsis kenyana is described for the first time. Pathogenicity tests revealed that Neopestalotiopsis mianyangensis, N. paeonia-suffruticosa, N. terricola, Pestalotiopsis kenyana, Seiridium guangyuanum, S. vernicola, and S. oleae are pathogenic on detached olive leaves.


Introduction
Woody oil plant is a general term for the fruits, leaves or seeds of woody plants that are used for oil extraction. It is a renewable resource for many oil products, such as edible oils, aromatic oils, industrial oils, and oils for bioenergy [1]. Woody oil plants have a long history of cultivation in China and are commonly grown in Sichuan, Yunnan, and Hunan Provinces [2]. About 8000 woody oil plant species are known in China, and over 300 of them contain above 20% fat content in fruits or seeds, such as Camellia oleifera, Sapium sebiferum, Vernicia fordii, etc. [3]. Statistical data from 2017 indicate that plantations of these plants produced 1,024,500 tons of oil annually (State-owned Forest Farms and Nurseries Station, State Forestry Administration of China, 2018).
To provide a stable molecular-based phylogeny to this morphologically highly similar fungal group, we have been studying pestalotiod taxa from woody oil plants in Sichuan Province, China. The study aimed to resolve pestalotiod taxa at the species level based on both morphological characteristics and multi-locus phylogenetic analyses while also testing the pathogenicity of these fungi on detached olive leaves.

Specimen Collection, Examination, and Fungal Isolation
Diseased branches and leaves of woody oil plants (Camellia oleifera, Olea europaea, Paeonia suffruticosa, Sapium sebiferum, and Vernicia fordii, Figure 1) were collected from Sichuan Province, China (detailed information of the collection sites are mentioned in Section 3.2). Specimens were taken to the laboratory and observed using a Motic SMZ 168 stereo microscope. Micro-morphological characteristics were observed and photographed with a Nikon ECLIPSE Ni compound microscope fitted with a Canon EOS 600D digital camera. Measurements were taken by Tarosoft Image Frame Work program v. 0.9.7 and the images for figures were processed with Adobe Photoshop CS6 Extended v. 13.0 software. Fungal isolates were obtained by the single spore isolation method. Germinated conidia were transferred aseptically to potato dextrose agar (PDA) plates and grown at 25 • C in daylight. Colony color [25] and other characters were observed and measured after two weeks.  [26]. The scientific names of the new taxa were registered in MycoBank (www.mycobank.com accessed on 25 August 2022)) and Facesoffungi [27].

PHI Analysis
PHI analysis (Pairwise Homoplasy Index) is used for confirming new species with low statistical support and significant tree lengths. The evidence of significant genetic recombination between the novel species and its closely related species was conducted (If Fw > 0.05).

Pathogenicity Tests on Olive Leaves
Olive (Olea europaea), widely cultivated as a woody oil plant in Sichuan Province, was selected as the material for the pathogenicity tests. Fresh and healthy leaves were collected from three-year-old olive trees in Chengdu olive cultivation base. Two experimental methods were established to check the infection ability and the virulence of the pestalotiodlike fungi on olive leaves. The olive leaves were washed thoroughly in running water and surface disinfected for 1 min in 75% alcohol, washed with sterilized double distilled water, and dried on sterile filter papers.
Unwounded method: three olive leaves were inoculated per fungal species. A mycelial plug (5 mm diam.), taken from the edge of a two weeks-old growing culture, was placed on the olive leaf. Another two olive leaves that served as the negative control were each inoculated with a sterile PDA plug. Wounded method: an artificial injury was made using a sterilized dissecting needle on the olive leaf surface and a 5 mm diam. A mycelial plug was placed over the wound. All leaves were placed in a humid chamber at 25 • C for 7 d.
Development of any disease symptoms were checked daily following inoculation, and the lesion length was measured after 7 d using a digital caliper. After 14 d, re-isolation was conducted from the margin of the necrotic tissue to recover the infected fungi and to meet Koch's postulates. Additionally, isolation from the negative controls was conducted to verify that no endophytic pestalotiod fungus was present.

Statistical Analysis
Data related to the pathogenicity test were analyzed with SPSS version 24 software (SPSS Inc., Chicago, IL, USA) by one-way variance analysis. The means were compared using Duncan's test at a significance level of p ≤ 0.05.

Phylogenetic Analyses
The first combined ITS, tub2, and tef1-α sequence dataset was analyzed to infer the interspecific relationships within Neopestalotiopsis. The concatenated data matrix consisted of sequences of 112 isolates, including the outgroup taxon Pestalotiopsis diversiseta (MFLUCC 12-0287). A total of 1826 characters including gaps (502 for ITS, 753 for tub2, and 571 for tef1-α) were included in the alignment. For the Bayesian inference, the HKY + I + G model with invgamma-distributed rate was selected for ITS, GTR + I + G model with invgammadistributed rate was selected for tub2 and the HKY + G model with gamma-distributed rate was selected for tef1-α. The maximum likelihood tree confirmed a similar tree topology to the Bayesian consensus tree, and the best-scoring ML tree is shown in Figure 2   Phylogram generated from RAxML analysis based on combined ITS, tef1-α, and tub2 sequence data of Neopestalotiopsis isolates. The tree was rooted to Pestalotiopsis diversiseta (MFLUCC 12-0287). The ML, MP bootstrap supports (≥60%) and BI posterior probabilities (≥0.90 PP) supports are given near the nodes, respectively. Isolates from this study are marked in red and ex-type strains are marked in bold.
Culture characteristics: Colonies on PDA reaching 20 mm diam. after 2 weeks at 25 • C are dense, circular, flattened to slightly raised, their surfaces are rough, radially furrowed at the centre, and smooth at the margin, with edge indented and velvety from above, whitish at the margin, pale yellowish at the centre, and from below, honey yellowish, producing yellowish pigmentation on agar medium.

Pathogenicity Assay
Results of pathogenicity tests were determined 7 d after inoculation. Seven species out of nine produced brown lesions showing virulence on wounded leaves. The isolates of Seiridium oleae were the most aggressive, causing significantly longer f brown lesions (average lesion length 41 mm, SD = 8.5), followed by S. guangyuanum (average lesion length 35 mm, SD = 5) (Figure 17b). Both Neopestalotiopsis and Pestalotiopsis species displayed average size lesions, while Seiridium brachiata and S. ceratosporum did not show significant pathogenicity on olive leaves. No symptoms were observed on leaves using the unwounded method (Figure 17a) nor in any control group. Koch's postulates were fulfilled by reisolating the same fungi and the colony and morphological characters were verified.

Discussion
Pestalotiod species have been extensively investigated on several woody oil plants globally, especially in Italy, Spain, and the USA [43][44][45], but no extensive studies have been carried out in China. We conducted wide-ranging surveys of pestalotiod fungi associated with diseased branches and leaves of commonly grown woody oil plants in Sichuan Province, resulting in 29 isolates. Based on multi-locus phylogeny and morphological analyses, six novel species (Neopestalotiopsis mianyangensis, N. paeonia-suffruticosa, N. terricola, Seiridium guangyuan, S. vernicola, and S. oleae) and three new records (Pestalotiopsis kenyana, Seiridium ceratosporum, and S. rosarum) were identified and described. Additionally, the sexual morph of Pestalotiopsis kenyana was reported for the first time in this study.
Conidial characters such as length, width, median cell length, the color of median cells, and length of the apical appendages are widely used for taxonomic purposes and inter-specific delineation of pestalotiod fungi. However, Maharachchikumbura et al. observed that some Pestalotiopsis species have similar conidial dimensions which cluster in distinct clades [13]. In this study, we observed that the conidial shape, size, and color of Neopestalotiopsis terricola isolated from Paeonia suffruticosa varied after inoculation on the olive leaves (Figures 7 and 8). We found that the conidia of N. paeonia-suffruticosa on PDA are shorter than those naturally found on woody plants ( Figure 7). Hence, we suggest that conidial length and width, even color, may not be a reliable taxonomic character alone for distinguishing Neopestalotiopsis species. Though the conidial appendages appear to be highly informative at the species level, the apical appendages vary in length, number, shape, branched or unbranched nature, presence or absence of knobbed tips, and the position of attachment to the conidial body [46]. Bonthond et al. [21] compared the appendages of most species in the genus Seiridium and found that many species have significant differences in the basal appendages. The same result was confirmed by our study.
Since pestalotiod species may have an endophytic, saprobic, or pathogenic lifestyle, we determined their pathogenicity on detached leaves of 3-year-old Olea europaea by inoculating colonized mycelial discs via two different methods ('wounded' and 'unwounded'). The results showed that all tested species were unable to provoke brown lesions on detached and unwounded olive leaves; however, most of the pestalotiod fungi were able to infect the host in the wounded method, revealing that injuries to woody oil plants could lead to fungal infection. Moreover, these isolates showed different virulence spectra with Seiridium oleae isolates being highly aggressive to detached olive leaves, whereas S. ceratosporum was much less aggressive on the same olive leaves. These results showed that pestalotiod fungi are pathogenic and responsible for causing diseases of commonly grown woody oil plants by fulfilling Koch's postulates.
It is worth noting that most pestalotiod species have a wide host range, and they have been reported to infect many economic plants resulting in severe diseases, e.g., avocado, blueberry [47], mango [48], strawberry [49], and kiwifruit [50]. As most pestalotiod species identified in this study were pathogenic to detached leaves of Olea europaea, they may pose potential threats to olive production by causing plant losses and reducing plant quality. The pathogenicity tests and host range studies indicated that pestalotiod fungi obtained from this study are not host-specific, and they can infect woody oil plants other than those from which they were initially isolated. This indicates that some Neopestalotiopsis, Pestalotiopsis, and Seiridium species can potentially infect a range of woody oil plants. The isolates were collected from different locations in the Sichuan Province, confirming the widespread distribution of the pathogen. Several biotic and abiotic factors, such as pest insects, hail, and many others, may cause injury to woody oil plants and could lead to fungal infection. To reduce the occurrence of diseases, the prevention of trunk wounds and the pruning of dead, dying, or diseased branches are likely to aid in reducing the incidence of disease.
Since woody oil plants are regarded as economical and commercial plant species in China, more extensive research on fungal pathogenic species from fresh material should be conducted to help clarify the pathogens and to verify the nature of the infection. This would contribute to potential intervention by taking preventive measures to reduce infection.

Informed Consent Statement:
The sequences data were submitted to GenBank.

Data Availability Statement:
The sequences data were submitted to GenBank.