Diversity of Monochaetia Species from Fagaceous Leaf Spots in China and Pathogenicity for Chinese Chestnut

ABSTRACT Pestalotioid fungi have been frequently studied with respect to their morphology, molecular phylogeny, and pathogenicity. Monochaetia is a pestalotioid genus that is morphologically characterized by 5-celled conidia with single apical and basal appendages. In the present study, fungal isolates were obtained from diseased leaves of Fagaceae hosts in China in 2016 to 2021 and identified based on morphology and phylogenetic analyses of the 5.8S nuclear ribosomal DNA gene with the two flanking internal transcribed spacer (ITS) regions, the nuclear ribosomal large subunit (LSU) region, the translation elongation factor 1-α (tef1) gene, and the β-tubulin (tub2) gene. As a result, five new species are proposed here, namely, Monochaetia hanzhongensis, Monochaetia lithocarpi, Monochaetia lithocarpicola, Monochaetia quercicola, and Monochaetia shaanxiensis. In addition, pathogenicity tests for these five species and Monochaetia castaneae from Castanea mollissima were conducted with detached leaves of Chinese chestnut. Results demonstrated that only M. castaneae successfully infected the host C. mollissima and caused brown lesions. IMPORTANCE Monochaetia is a pestalotioid genus, with members that are commonly known as leaf pathogens or saprobes; some strains were isolated from air, in which case their natural substrate is so far unknown. Fagaceae represents an ecologically and economically important plant family that is widely distributed in the Northern Hemisphere, including an important tree crop species, Castanea mollissima, which is widely cultivated in China. In the present study, diseased leaves of Fagaceae in China were investigated, and five new Monochaetia species were introduced based on morphology and phylogeny of combined ITS, LSU, tef1, and tub2 loci. Additionally, six species of Monochaetia were inoculated onto healthy leaves of the crop host Castanea mollissima to test their pathogenicity. The present study provides significant data on the species diversity, taxonomy, and host range of Monochaetia and enhances our understanding of leaf diseases of Fagaceae hosts.


DISCUSSION
In the present study, 9 isolates from Fagaceae leaf spots in China were identified as 5 new species of Monochaetia. However, we investigated only 5 Fagaceae hosts of more than 320 reported species in China, indicating that many hidden Monochaetia species may remain to be discovered from Fagaceae in the future.
Species of Monochaetia are commonly known as leaf pathogens or saprobes and are sometimes isolated from air (4, 13 to 15). In previous studies, 8 species were recorded from fagaceous hosts, inhabiting leaves of Castanea or Quercus (4,13,15). Of these, 3 species (Monochaetia bicornis, Monochaetia concentrica, and Monochaetia hysteriiformis) have not yet been sequenced. In the present study, 5 additional new species were revealed from Fagaceae based on conidial characteristics and molecular phylogenetic evidence (Table 1). Two new species, Monochaetia lithocarpi and M. lithocarpicola, were discovered from Lithocarpus, which represents a new host genus for Monochaetia. However, no Monochaetia species are known from the residual 5 genera of Fagaceae; considering that leaf spot pathogens of these are still poorly studied in most areas, it is possible that additional Monochaetia species may be revealed from them.
As shown in Table 1, Monochaetia concentrica and M. kansensis can inhabit both Castanea and Quercus (15). Therefore, while host association may be indicative for some species, it cannot be generally considered a reliable characteristic to separate Monochaetia species. Most Monochaetia species sequenced are still known from only a few confirmed isolates from a few localities and, as with other genera of Sporocadaceae, the possibility that many species have wider host ranges than the few published records may suggest cannot be excluded.
Conidial morphology, including pigmentation, septation and wall ornamentation of wall cells, position of appendages with respect to the apical and basal cells, and the number and branching pattern of apical appendages, has been widely used to separate pestalotioid genera and species (10, 11, 13). In the present study, we discovered the first species having nearly hyaline to pale brown conidia in Monochaetia, M. lithocarpicola (Fig. 5). This suggests that pigmentation might not be a robust characteristic to distinguish genera in Sporocadaceae, which was similarly observed for Allelochaeta (20).
This study revealed five new Monochaetia species associated with Fagaceae leaf spot disease symptoms in China. Because Castanea mollissima is a widely cultivated fagaceous host in China (4), we tested the pathogenicity of M. castaneae (from Castanea mollissima), M. hanzhongensis (from Quercus variabilis), M. lithocarpi (from Lithocarpus glaber), M. lithocarpicola (from L. glaber), M. quercicola (from Quercus acutissima and Q. aliena) and M. shaanxiensis (from Quercus baronii) to healthy leaves of C. mollissima. Only M. castaneae successfully infected C. mollissima and caused brown lesions, which implied that the other five fungal species are not pathogenic to the important crop fagaceous species C. mollissima.

MATERIALS AND METHODS
Sample collection and isolation. Investigations were conducted in Guangdong, Henan, and Shaanxi Provinces in China from 2016 to 2021 to collect diseased fagaceous leaf samples. A total of two host genera and five species in Fagaceae, namely, Lithocarpus glaber, Q. variabilis, Q. aliena, Q. acutissima, and Q. baronii, were investigated in the present study. The leaf samples were packed in paper bags and transferred to the laboratory for fungal isolation.
The diseased leaf samples were surface sterilized for 1 min in 75% ethanol, 3 min in 1.25% sodium hypochlorite, and 1 min in 75% ethanol, rinsed for 2 min in distilled water, and blotted on dry sterile filter paper. Then the diseased areas of the leaves were cut into pieces (0.5 by 0.5 cm) with an aseptic razor blade, transferred onto the surface of both PDA (200 g/L potato, 20 g/L dextrose, 20 g/L agar) and MEA (30 g/L malt extract, 5 g/L mycological peptone, 15 g/L agar) plates, and incubated at 25°C to obtain fungal hyphae. Hyphal tips were then removed to new PDA and MEA plates to obtain pure cultures. The cultures were deposited in the China Forestry Culture Collection Center (CFCC) (http://cfcc.caf.ac.cn/) and the specimens in the herbarium of the Chinese Academy of Forestry (CAF) (http://museum.caf.ac.cn).
DNA extraction, sequencing, and phylogenetic analyses. Genomic DNA was extracted from colonies grown on cellophane-covered PDA plates using a cetyltrimethylammonium bromide (CTAB) method (21). DNA quality was estimated by electrophoresis in 1% agarose gels, and the quality was measured with a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) following the user manual. The following primer pairs were used for amplification of the gene regions sequenced in the present study: ITS1/ITS4 for the 5.8S nuclear ribosomal DNA gene with the two flanking ITS regions (ITS1 and ITS2) (22), LR0R/LR5 for the nuclear ribosomal LSU region (23), EF1-728F/EF2 for the translation elongation factor 1-a (tef1) gene (24,25), and Bt2a/Bt2b for the b-tubulin (tub2) gene (26). The PCR conditions were set as follows: an initial denaturation step of 5 min at 94°C, 35 cycles of 30 s at 94°C, 50 s at 52°C (ITS and LSU) or 54°C (tef1 and tub2), and 1 min at 72°C, and a final elongation step of 7 min at 72°C. PCR amplification products were checked via electrophoresis in 2% agarose gels. DNA sequencing was performed using an ABI Prism 3730XL DNA Analyzer with a BigDye Terminator kit v.3.1 (Invitrogen, USA) at the Shanghai Invitrogen Biological Technology Company Ltd. (Beijing, China).
The quality of the amplified nucleotide sequences was checked and the sequences were assembled using SeqMan v.7.1.0. Reference sequences were retrieved from the National Center for Biotechnology Information (NCBI) database. Sequences were aligned using MAFFT v.7 (http://mafft.cbrc.jp/alignment/server) (27) and corrected manually using MEGA 6 (28). The nucleotide sequence data from the present study were deposited in GenBank, and the accession numbers are listed in Table 2. The phylogenetic analyses of the combined matrices were performed using ML and BI methods. ML analysis was implemented with the CIPRES Science Gateway portal (https://www.phylo.org) using RAxML-HPC BlackBox v.8.2.10 (29), employing a GTRGAMMA substitution model with 1,000 bootstrap replicates, while BI analysis was performed using a Markov chain Monte Carlo (MCMC) algorithm in MrBayes v.3.0 (30). Two MCMC chains were run, starting from random trees, for 1,000,000 generations, and trees were sampled every 100th generation, resulting in a total of 10,000 trees. The first 25% of trees were discarded as burn-in for each analysis. Branches with significant Bayesian posterior probabilities (BPPs) were estimated in the remaining 7,500 trees. Phylogenetic trees were viewed with FigTree v.1.3.1 and graphically processed with Adobe Illustrator CS5.
For closely related species with similar morphology, ITS, LSU, tef1, and tub2 sequences of the species were pairwise compared. For this, the sequences of species pairs were aligned, and the parts containing leading/trailing gaps were removed. Sequence differences of this alignment are recorded in the following way: number of nucleotide substitutions (excluding insertions and gaps)/total number of nucleotide characters, percentage of sequence substitutions, and numbers of insertions and gaps.
Morphology. The morphological data for the isolates collected in the present study were obtained from sporulating pure cultures grown on PDA or MEA plates in the dark at 25°C. The conidiomata were observed and photographed using a dissecting microscope (M205 C; Leica, Wetzlar, Germany). Microscope slides of conidiogenous cells and conidia were prepared in tap water, and the slides were examined and photographed with an Axio Imager 2 microscope (Zeiss, Oberkochen, Germany) equipped with an Axiocam 506 color camera or an Eclipse 80i microscope (Nikon, Tokyo, Japan) equipped with a Nikon digital sight DS-Ri2 camera, using differential interference contrast (DIC) illumination. For measurements, 50 conidia were randomly selected. Measurements of the conidia are reported as maximum and minimum (in parentheses) and the range representing the mean 6 SD of the number of measurements indicated. Culture characteristics were recorded from 9-cm PDA or MEA plates after 10 days of incubation at 25°C in the dark. To enable comparison of species growing on fagaceous hosts, measurement data and sequence data available are summarized in Table 1.
Pathogenicity. The isolates representing Monochaetia castaneae (CFCC 54354), M. hanzhongensis (CFCC 54451), M. lithocarpi (CFCC 54402), M. lithocarpicola (CFCC 54509), M. quercicola (CFCC 55515), and M. shaanxiensis (CFCC 54419) were selected for inoculations. Healthy leaves of Castanea mollissima were washed in distilled water, surface sterilized in 75% ethanol for 1 min, rinsed in distilled water, and then surface wounded with a sterile needle in both the left and right portions of the leaves. Conidia of the six Monochaetia species were harvested from 4-week-old PDA plates with 10 mL of sterilized water, and the conidial suspension was filtered through two layers of cheesecloth to eliminate debris and mycelia. The conidial suspension was adjusted to a final inoculum concentration of 1 Â 10 6 conidia/mL with sterile deionized water. Then, 10 mL of conidial suspension was placed in the left portion of the leaves; sterile water inoculated in the right portion served as the negative control. Each treatment had five replicates, and the experiment was carried out twice. The inoculated leaves were placed in transparent plastic bags at 25°C and .90% humidity in the dark for 10 days. After the appearance of symptoms, fungal isolates were reisolated from the infected leaves and identified based on the morphological and phylogenetic analyses to fulfill Koch's postulates.