Morphology, Phylogeny, and Pathogenicity of Pestalotioid Species on Camellia oleifera in China

Tea-oil tree (Camellia oleifera) is an important edible oil woody plant with a planting area of over 3,800,000 hectares in southern China. Pestalotioid fungi are associated with a wide variety of plants worldwide along with endophytes, pathogens, and saprobes. In this study, symptomatic leaves of C. oleifera were collected from Guangdong, Guangxi, Hainan, Hunan, and Jiangsu Provinces and pestalotioid fungi are characterized based on combined sequence data analyses of internal transcribed spacer (ITS), beta tubulin (tub2), and translation elongation factor 1-alpha (tef-1α) coupled with morphological characteristics. As a result, seven species were confirmed, of which five species are described as new viz. N. camelliae-oleiferae, P. camelliae-oleiferae, P. hunanensis, P. nanjingensis, P. nanningensis, while the other two are reported as known species, viz., N. cubana and N. iberica. Pathogenicity assays showed that all species except for P. nanjingensis developed brown lesions on healthy leaves and P. camelliae-oleiferae showed stronger virulence.


Introduction
Tea-oil tree (Camellia oleifera Abel.) is a unique woody edible oil species in China, mainly distributed in the Qinling-Huaihe River area. It has a long history of cultivation and utilization for more than 2300 years since ancient China [1]. Statistical data for 2014 indicated that these plantations comprise over 3,800,000 hectares and produce 518,000 tons of edible oil (State-owned Forest Farms and Nurseries Station, State Forestry Administration of China, 2016). Camellia oil, obtained from C. oleifera seeds, is rich in unsaturated fatty acids and unique flavors, and has become a rising high-quality edible vegetable oil in China [2]. Thus, the development of the C. oleifera industry is of great significance for the national economy and poverty alleviation of local farmers in China.
The expanding cultivation of C. oleifera over the last several decades has also attracted increasing attention from plant pathologists to infectious diseases on this crop. Anthracnose disease caused by Colletotrichum species is one of the foremost diseases in southern China, which can infect leaves and fruits of C. oleifera, causing up to a 40% fruit drop and up to 40% camellia seeds loss [3]. Several studies have focused on the diversity and the pathogenicity of fungi in this special habitat [3][4][5]. However, relatively little is known about the taxonomy, genetic diversity, and pathogenicity of pestalotioid species on C. oleifera.
Pestalotioid species represent a cosmopolitan group of fungi occupying diverse ecological behavior as plant pathogens, endophytes, or saprobes, and are widely distributed throughout tropical and temperate regions [6][7][8]. However, species identification in this

Pathogenicity Testing
Young and healthy leaves of Camellia oleifera were collected from trees growing in the greenhouse. The leaves were washed with tap water, then submerged in 70% ethanol for 2 min, and finally rinsed in sterilized water twice. The petioles of leaves were wrapped with damp cotton wool and the leaves were placed into petri dishes, three leaves per dish. One piercing wounds of each leaf were made in the mid-region forming a tiny little dot using a sterilized needle. Three drops of 6 µL spore suspension (10 6 conidia/mL) were individually placed directly onto the leaf upper surfaces. For the control group, 6 µL of sterilized water was used. Each set of three leaves per petri dish was incubated with a different isolate. The petri dishes were placed inside a plastic box and the leaves incubated at 25 • C with humidity and 12/12 h fluorescent light/dark cycle. After 5 d, the leaves were examined for symptom development, and the diameter of diseased spot was measured.

Phylogenetic Analyses
The first sequence datasets for the ITS, tef-1α and tub2, were analyzed in combination to infer the interspecific relationships within Neopestalotiopsis. The combined species phylogeny of the Neopestalotiopsis isolates consisted of 105 sequences, including the outgroup Pestalotiopsis trachicarpicola (culture OP068). A total of 1389 characters including gaps (479 for ITS, 498 for tef-1α, and 412 for tub2) were included in the phylogenetic analysis.
Similar tree topologies were obtained by ML and BI methods, and the best scoring ML tree is shown in Figure 1. ML bootstrap values and BI posterior probabilities (MLBS/BIPP) are given at nodes of the phylogram (Figure 1). The phylogenetic tree inferred from the concatenated alignment resolved the ten Neopestalotiopsis isolates from symptomatic leaves of Camellia oleifera into four well-supported monophyletic clades that represent one novel species, one undetermined species and two known species of Neopestalotiopsis ( Figure 1).   The second sequence datasets for the ITS, tef-1α and tub2 were analyzed in combination to infer the interspecific relationships within Pestalotiopsis. The combined species phylogeny of the Pestalotiopsis isolates consisted of 129 sequences, including the outgroup Neopestalotiopsis magna (culture MFLUCC 12-652). A total of 1557 characters including gaps (515 for ITS, 537 for tef-1α, and 505 for tub2) were included in the phylogenetic analysis. Similar tree topologies were obtained by ML and BI methods, and the best scoring ML tree is shown in Figure 2. ML bootstrap values and BI posterior probabilities (MLBS/BIPP) are given at nodes of the phylogram (Figure 2). The phylogenetic tree inferred from the concatenated alignment resolved the 12 Pestalotiopsis isolates from symptomatic leaves of Camellia oleifera into four well-supported monophyletic clades that represent four novel species of Pestalotiopsis (Figure 2).   Isolates from this study are marked in red and the identified species is marked in yellow. Ex-type strains are labeled with *.
Culture characteristics: Colonies on PDA reaching 55 mm diameter after seven days at 25 • C. Colonies filamentous to circular, with dense aerial mycelium on surface, fruiting bodies black.
Culture characteristics: Colonies on PDA reaching 70 mm diameter after seven days at 25 • C. Colonies filamentous to circular, medium dense, aerial mycelium on surface flat or raised, pycnidia abundant, fruiting bodies black.
Notes: Neopestalotiopsis cubana was originally described from leaf litter in Cuba [8]. In the present study, two isolates from leaves of symptomatic C. oleifera were congruent with N. cubana based on morphology and DNA sequences data (Figure 1). We therefore describe N. cubana as a known species for this clade. Notes: Neopestalotiopsis cubana was originally described from leaf litter in Cuba [8]. In the present study, two isolates from leaves of symptomatic C. oleifera were congruent with N. cubana based on morphology and DNA sequences data (Figure 1). We therefore describe N. cubana as a known species for this clade.  Culture characteristics: Colonies on PDA reaching 70 mm diameter after seven days at 25 • C. Colonies filamentous to circular, medium dense, aerial mycelium on surface flat or raised, with filiform margin, fluffy, fruiting bodies black.
Notes: Neopestalotiopsis iberica was originally described from leaves and stems of Eucalyptus globulus in Portugal [30]. In the present study, three isolates from leaves of symptomatic C. oleifera were congruent with N. iberica based on morphology and DNA sequences data (Figure 1). We therefore describe N. iberica as a known species for this clade.
Notes: Neopestalotiopsis iberica was originally described from leaves and stems of Eucalyptus globulus in Portugal [30]. In the present study, three isolates from leaves of symptomatic C. oleifera were congruent with N. iberica based on morphology and DNA sequences data (Figure 1). We therefore describe N. iberica as a known species for this clade. Culture characteristics: Colonies on PDA reaching 70 mm diameter after seven days at 25 °C. Colonies filamentous to circular, medium dense, with white sparse mycelium, fruiting bodies black.
Culture characteristics: Colonies on PDA reaching 70 mm diameter after seven days at 25 • C. Colonies filamentous to circular, medium dense, with white sparse mycelium, fruiting bodies black.
Culture characteristics: Colonies on PDA reaching 50 mm diameter after seven days at 25 °C. Colonies filamentous to circular, with sparse aerial mycelium, fruiting bodies black.
Culture characteristics: Colonies on PDA reaching 50 mm diameter after seven days at 25 • C. Colonies filamentous to circular, with sparse aerial mycelium, fruiting bodies black.
Culture characteristics: Colonies on PDA reaching 60 mm diameter after seven days at 25 °C. Colonies filamentous to circular, medium dense, aerial mycelium on surface flat, fruiting bodies black.
Culture characteristics: Colonies on PDA reaching 80 mm diameter after seven days at 25 °C. Colonies filamentous to circular, medium dense, white aerial mycelium on surface flat or raised.
Culture characteristics: Colonies on PDA reaching 80 mm diameter after seven days at 25 • C. Colonies filamentous to circular, medium dense, white aerial mycelium on surface flat or raised.

Pathogenicity Assay
After five days, for the pathogenicity tests, N. camelliae-oleiferae, N. cubana, N. iberica Neopestalotiopsis sp.1, P. camelliae-oleiferae, P. hunanensis, and P. nanningensis developed brown lesions on wounded leaves (right), whereas the controls showed no symptoms (left). Neopestalotiopsis sp.1 had the highest virulence, while P. nanjingensis did not cause obvious symptoms ( Figure 10). Koch's postulates were fulfilled by reisolating the same fungi and verifying its colony and morphological characters.

Pathogenicity Assay
After five days, for the pathogenicity tests, N. camelliae-oleiferae, N. cubana, N. iberica Neopestalotiopsis sp.1, P. camelliae-oleiferae, P. hunanensis , and P. nanningensis developed brown lesions on wounded leaves (right), whereas the controls showed no symptoms (left). Neopestalotiopsis sp.1 had the highest virulence, while P. nanjingensis did not cause obvious symptoms ( Figure 10). Koch's postulates were fulfilled by reisolating the same fungi and verifying its colony and morphological characters.

Discussion
In this study, an investigation of C. oleifera diseases in China was carried out and Camellia leaf disease caused by pestalotioid fungi was observed as a common disease. Identification of our collections was conducted, based on isolates from symptomatic leaves of C. oleifera using three combined loci (ITS, tef-1α and tub2), as well as morphological characteristics. It includes N. cubana, N. iberica, as well as five new species named N. camelliae-oleiferae, P. camelliae-oleiferae, P. hunanensis, P. nanjingensis, and P. nanningensis.
The expanding cultivation of C. oleifera over the last several decades has attracted increasing attention from plant pathologists to infectious diseases on this crop. Therein, pestalotioid species are more frequently regarded as endophytes or latent pathogens caus-

Discussion
In this study, an investigation of C. oleifera diseases in China was carried out and Camellia leaf disease caused by pestalotioid fungi was observed as a common disease. Identification of our collections was conducted, based on isolates from symptomatic leaves of C. oleifera using three combined loci (ITS, tef-1α and tub2), as well as morphological characteristics. It includes N. cubana, N. iberica, as well as five new species named N. camelliae-oleiferae, P. camelliae-oleiferae, P. hunanensis, P. nanjingensis, and P. nanningensis.
The expanding cultivation of C. oleifera over the last several decades has attracted increasing attention from plant pathologists to infectious diseases on this crop. Therein, pestalotioid species are more frequently regarded as endophytes or latent pathogens causing diseases only on specific situations [4,6,12,63,64]. Understanding the diversity of pestalotioid species and the genetic variation within pathogen populations could help in developing sustainable disease management strategies.
Pestalotioid fungi (Pestalotiopsidaceae, Sordariomycetes) are species-rich asexual taxa, which are common pathogens that cause a variety of diseases, including leaf spots, shoot dieback, fruit rots and various post-harvest diseases [6,8,15,19,20,46,65]. As many peatalotioid species have overlapping morphological traits, sequence data is essential to resolve these three genera and introduce new species [8]. Combined gene sequence of ITS, tef-1α, and tub2 can provide a better resolution for Pestalotiopsis and Pseudopestalotiopsis. However, more genes are needed to provide better resolution and support in Neopestalotiopsis. Furthermore, this is the first systematic report of Neopestalotiopsis and Pestalotiopsis fungi associated with Camellia oleifera in China, which indicates that there may be a high undescribed diversity of fungi in this host.
Pathogenicity tests of eight pestalotioid species from Camellia oleifera showed that all species except for P. nanjingensis were capable of infecting wounded leaves. Neopestalotiopsis sp.1 and P. camelliae-oleiferae showed stronger virulence, with lesion diameters ranged from 14.7 to 17.8 mm on leaves of the Neopestalotiopsis sp.1 isolate (CSUFTCC61) and 13.5 to 15.5 mm on leaves of the P. camelliae-oleiferae isolate (CSUFTCC08). All pathogenicity tests were performed with a single C. camellia cultivar. Since different C. oleifera cultivars may have different resistance to pestalotioid species, more cultivars of C. oleifera should be studied for the variation of their resistance to pestalotioid pathogens. During the tests, the symptoms vary considerably with factors, such as relative humidity, temperature, and the inoculum concentration. In the future, field conditions with natural inoculum should be conducted rather than just in vitro artificial inoculation.

Conclusions
Seven peatalotioid species (two known species and five new species) were described and illustrated. This is the first systematic report of Neopestalotiopsis and Pestalotiopsis fungi associated with Camellia oleifera in China. The pathogenicity of these species on leaves were examined and showed that there were significant differences in the pathogenicity. Data Availability Statement: All sequence data are available in NCBI GenBank following the accession numbers in the manuscript.