Biodiversity Data Journal :
Short Communication
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Corresponding author: Dan Li (lidan@jlau.edu.cn), Changtian Li (lct@jlau.edu.cn), Yu Li (yuli966@126.com)
Academic editor: Renan Barbosa
Received: 13 Jun 2022 | Accepted: 28 Aug 2022 | Published: 11 Oct 2022
© 2022 Xiaoya An, Guohui Cheng, Hanxing Gao, Yang Yang, Dan Li, Changtian Li, Yu Li
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
An X, Cheng G, Gao H, Yang Y, Li D, Li C, Li Y (2022) First report of Cladobotryum verticillatum (Ascomycota, Hypocreaceae) causing cobweb disease on Paxillus involutus. Biodiversity Data Journal 10: e87697. https://doi.org/10.3897/BDJ.10.e87697
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Paxillus, a type of ectomycorrhizal fungi distributed widely in the world, is also an essential category for researching bioactive substances and pharmacological functions. We discovered fruitbodies of Paxillus involutus covered in a layer of white mycelium in 2020. Cladobotryum verticillatum, a pathogenic fungus related to cobweb disease, was isolated and identified based on morphological and phylogenetic features. Koch's postulates were used to confirm the pathogenicity. The host range test revealed that C. verticillatum could cause disease in all examined mushrooms except Ganoderma sichuanense. To our knowledge, C. verticillatum is a new record species in China and a new pathogen on Paxillus involutus.
Paxillaceae, Hypocreaceae, mycoparasite, cobweb disease
Paxillus Fr. is a genus in the family Paxillaceae, order Boletes. Its members form typical ectomycorrhizal structures with a variety of wooden hosts (
Mycoparasites are an important ecological category that interacts with other fungi (including parasites and saprobes) (
In August 2020, we discovered Paxillus involutus basidiocarps covered with a layer of white mycelium in the Changbai Mountain Biosphere Reserve (CMBR), Jilin Province, China. Broad-leaved forests with Quercus mongolica and Betula platyphylla as the primary tree species supported the diseased fruitbodies. Crippled and decaying mushrooms were collected (
Diseased fruitbodies were cut into small pieces (5 mm × 5 mm × 5 mm) with a sterilised scalpel, and infected tissues were immersed in 75% ethanol solution for 45 s before being rinsed three times with sterilised water. Then, dried surface with sterile filter paper, placed on Potato Dextrose Agar (PDA) plates containing 100 mg/l streptomycin sulphate, incubated at room temperature, and transferred the culture to fresh PDA plates when the fungal hyphae emerged and cultured the plates at 25°C for five days to allow the colonies to sporulate fully. Use the single spore separation to get the pure cultures following the method described by
After activating the pathogen, picked some hyphae with the inoculation needle from the culture and transferred them on a slide aseptically for morphological identification. Mycelial samples with conidiophores and conidia were observed under a Zeiss Axio Lab A1 light microscope (Carl Zeiss, Germany) and microscopic observations made with objectives of 10x, 20x, 40x and 100x oil immersion. All measurements and photographs were performed using a Zeiss Imager A2 microscope with an Axiocam 506 colour camera and integrated software. Microscopically, the characteristics of 30 conidia and conidiophores from the isolates were observed. Morphological identification was performed using the
The genomic DNA of the pathogen (C. verticillatum) was extracted from the mycelia of colonies on PDA. Gene sequences of ITS, TEF1 and RPB2 were amplified by a polymerase chain reaction (PCR) with the primer pairs of ITS4/ITS5 (
BLASTn searches with the sequences were performed against NCBI to detect the most closely-related species (http://www.blast.ncbi.nlm.nih.gov/). Phylogenetic trees were constructed using ITS, TEF1 and RPB2 sequences, and phylogenetic analyses were performed with the Maximum Likelihood (ML) and Maximum Parsimony (MP) methods. Multiple alignments of all present sequences were automatically generated using MAFFT V. 7.471, and manual improvements were made using BioEdit when necessary (
The experiments were carried out in duplicate to confirm the pathogenicity of the strain YW, according to Koch's postulates. We found Pa. involutus fruiting bodies in a birch forest on the campus of Jilin Agricultural University and inoculated them with spore suspension (50 μl) on caps. We observed the process in the wild and recorded changes in disease symptoms for ten days. Select the fruiting body with white mycelium for fungal isolation. Furthermore, the host range tests were investigated by inoculating it on to nine commercial mushroom species: Pleurotus ostreatus, Hypsizygus marmoreus, Agrocybe aegerita, Pleurotus geesteranus, Pleurotus citrinopileatus, Flammulina filiformis, Pleurotus salmoneostramineus, Ganoderma sichuanense and Agaricus bisporus. All mushrooms were grown on the substrate and kept in the growing station. Mushrooms were inoculated with one droplet (50 μl) of spore suspension (5 × 106 unit/ml) mixed with Tween 80 on the upper surface of caps when they reached 3 to 4 cm in diameter (Pl. ostreatus, Pl. salmoneostramineus, Pl. geesteranus, Pl. citrinopileatus, Aga. bisporus, Hyps. marmoreus, Agr. aegerita) or stipe (F. filiformis). For G. sichuanense, inoculated the spore suspension on the solid layer under the pileus. Placed all mushroom bags at 25℃ and kept the air humidity at 80%–90%. The incident was observed and photographed.
Colonies spread, appearing fluffy, lanose, tufted or fine linen, white, with suberect tufts about 1–2 cm high, at length sinking and fading. Mycelium is branched, septate and hyaline with rich inclusions. Hyaline conidiophores have one to three septa, are verticillately or irregularly branched and their carriers are branched into two to five phialides. Conidia are 10.6–16.2 × 6.6–11.1 µm, one-celled, smooth- and thin-walled, hyaline, elliptical or elliptical-oblong, with protruding basal scars (Fig.
Field symptoms and morphological characteristics of Cladobotryum verticillatum. A Diseased fruiting bodies in the wild; B Colony on PDA; C Conidiophores with whorled and single phialides; D-F Tapered conidiogenous cells form singly or in whorls; G Conidiogenous cells; H Conidia; I, J chlamydospore; K, L Healthy mushrooms in the wild; M-P Mushrooms artificially inoculated with pathogens at 24 h, 48 h, 72 h and seven days, respectively. Bars: C–G, I, J = 20 μm; H = 10 μm
The BLAST results showed that the ITS sequence of strain YW was 99.83% similar to MT237489, the RPB2 sequence was 99.72% similar to FN868678, and the TEF1 sequence was 99.02% similar to FN868742, respectively. The dataset for phylogenetic analyses contained 27 ITS sequences, representing 19 species, choosing Trichoderma virid as the outgroup taxon. Multi-locus data were concatenated, which comprised 2554 characters with ITS 597 characters, TEF1 888 characters and RPB2 1069 characters. Estimated base frequencies were as follows: A = 0.233413, C = 0.296140, G = 0.248170 and T = 0.222278; substitution rates AC = 1.489328, AG = 3.647092, AT = 1.111646, CG = 0.925803, CT = 7.920581 and GT = 1.000000. In the resulting tree (Fig.
The pathogenicity test revealed that all the inoculated Pa. involutus exhibited first symptoms after 24 hours, with taupe lesions appearing on the surface of the gills (Fig.
The strain YW was tested on nine commercial mushroom types and found to be capable of infecting all but G. sichuanese. After inoculating the spore suspension on the stipes or caps of fruiting bodies, the hyphae began to grow. Typical cobweb signs, such as small brown spots, were seen 1–3 days post-inoculation (dpi). The white mycelia were then visible, and the fruiting bodies were rotting and covered in massive spores after 3-5 days. Finally, the mushrooms wilted and rotted, mirroring the characteristics of the field sample (Fig.
Disease development on different mushrooms after inoculating Cladobotryum verticillatum. A–D Pictures of Hypsizygus marmoreus in healthy condition, 1 dpi, 2 dpi and 3 dpi; E–H Pictures of Agrocybe aegerita in health, 1 dpi, 2 dpi and 3 dpi; I–L Pictures of Flammulina filiformis in health, 1 dpi, 2 dpi and 4 dpi; M–P Pictures of Pleurotus ostreatus in health, 1 dpi, 2 dpi and 8 dpi; Q–T Pictures of Pleurotus salmoneostramineus in health, 1 dpi, 2 dpi and 7 dpi; U–X Pictures of Pleurotus citrinopileatus in health, 1 dpi, 2 dpi and 3 dpi; Y–b Pictures of Pleurotus geesteranus in health, 1 dpi, 2 dpi and 3 dpi; C–f Pictures of Agaricus bisporus in health, 1 dpi, 2 dpi and 3 dpi.
Disease processions on F. filiformis, Aga. bisporus, Agr. aegerita, Hyps. marmoreus and Pl. citrinopileatus, were usually completed within four days and caused serious damage. White hyphae were visible on the first day post-inoculation and spread quickly, causing the mushrooms to become soft and brown. Symptoms of Aga. bisporus and Hyp. marmoreus were similar, with brown spots and mycelia visible at the inoculation site. Mycelia eventually covered the cap of Aga. bisporus and spread to the stalk-cap junction, resembling a spider's web. Pleurotus citrinopileatus, unlike others, had no brown spots on the cap, but as the hardness decreased or even disappeared, it eventually fell in clusters and turned brown.
Although Pl. ostreatus, Pl. salmoneostramineus and Pl. geesteranus displayed symptoms earlier, the progression was slow and prolonged. The hyphae grew on the first day, but no lesions were visible. Hyphae continued to stretch, causing the mushrooms to stop growing and atrophy, and the surface of the fruitbodies to become covered in white mycelia. When inoculated on the primordium or cap, G. sichuanense showed less sensitivity or high resistance to the pathogen when compared to other edible fungi.
Based on morphological and molecular characteristics, we isolated a fungal pathogen from diseased Pa. involutus and identified it as C. verticillatum. It was originally described by Heinrich and named by
Hypomyces/Cladobotryum species that live on Polyporales may have a lower host-selection than others (
We thank the Engineering Research Center of Edible and Medicinal Fungi, Ministry of Education, Jilin Agricultural University, China, for providing the laboratory and equipment for the duration of this project. We thank Lan Yao and Jianhua Lv from Jilin Agricultural University for the sample collection. We thank Yang Wang at Shenyang Agricultural University for his help in the process of mushroom hunting for supplementary experiments.
(i) U20A2046 funded by the National Natural Science Foundation of China, (ii) the earmarked fund for CARS-20 (Edible Mushroom) funded by the Ministry of Agriculture and Rural Affairs, PRC, (iii) 1630042022003 funded by the Ministry of Finance of China and (iv) 322QN365 funded by the Natural Science Foundation of Hainan Province, China.
Xiaoya An did the experiment, analysed the data and wrote the manuscript. Guohui Cheng, Hanxing Gao and Yang Yang collected the sample, isolated the fungi and performed the phylogeny analysis. Dan Li, Changtian Li and Yu Li conceived and coordinated the study. All authors contributed critically to the drafts and gave final approval for publication.
The authors declared that they have no conflict of interest.
Accession numbers include details such as locality, isolate numbers of the sequences used for this study