Diversity of Fungi Isolated from Potato Nematode Cysts in Guizhou Province, China

Potatoes rank third in terms of human consumption after rice and wheat. Globodera spp. are significant pests of potato crop worldwide. Globodera rostochiensis was found in Weining County, Guizhou Province, China, in 2019. We collected soil from the rhizosphere zone from infected potato plants and separated mature cysts through simple floatation and sieving methods. The selected cysts were surface-sterilized, and the colonized fungi were isolated and purified. At the same time, the preliminary identification of fungi and fungi parasites on the cysts of nematodes was carried out. This study aimed to define the species and frequency of fungi-colonizing cysts of G. rostochiensis collected from Weining County, Guizhou Province, China, and provide a basis for the control of G. rostochiensis. As a result, 139 strains of colonized fungi were successfully isolated. Multigene analyses showed that these isolates included 11 orders, 17 families, and 23 genera. The genera Fusarium (with a separation frequency of 59%), Penicillium (11%), Edenia (3.6%), and Paraphaeosphaeria (3.6%) were the most frequently occurring. Among the 44 strains, 27 had a colonization rate of 100% on the cysts of G. rostochiensis. Meanwhile, the functional annotation of 23 genera indicated that some fungi have multitrophic lifestyles combining endophytic, pathogenic, and saprophytic behavior. In conclusion, this study showed the species composition and lifestyle diversity of colonized fungi from G. rostochiensis and demonstrated these isolates as potential sources of biocontrol agents. Colonized fungi were isolated from G. rostochiensis for the first time in China, and the taxonomic diversity of fungi from G. rostochiensis was clarified.


Introduction
The potato is one of the most widely grown staple foods [1]. Potatoes provide more calories, protein, and minerals than any other staple crop [2]. As populations grow and urbanization intensifies, potato production has surged due to increased global consumption [3]. However, production is still adversely affected by pests and pathogens, including the potato cyst nematodes (PCNs) Globodera rostochiensis and G. pallida [4]. Both species contain pathotypes, and some closely related and very similar species are of minor economic importance.
PCNs-Globodera spp.-are among the most significant pests of potato crops worldwide [5]. PCN species are believed to have evolved in South America but now have a worldwide distribution and can be major and persistent pests except in the warmest soils [6]. The life cycle of PCNs is well-adapted to the host, and they can survive in various environments. Root exudates from Solanaceae activate juveniles, which can cause up to 80% of the nematodes to hatch under suitable environmental conditions [7]. Yield loss due to PCNs has been reported as 90-100% in Europe and North America [8]. In India, up to Bursaphelenchus xylophilus, and Ditylenchus destructor) [40]. In summary, due to various geographical locations, the diversity of colonizing fungi in the soil, and planting methods, there are significant differences in colonizing fungi strains and dominant populations in regions where G. rostochiensis occurs, and these colonizing fungi often have different degrees of nematicidal activity. The products developed from it have been successfully applied to controlling G. rostochiensis [41,42].
In China, fungal antagonists of PCNs have not been investigated so far due to G. rostochiensis only being found in 2018. Thus, an effective technique is necessary to suppress the PCN population in Weining and prevent its spread. In addition, interactions between microorganisms and G. rostochiensis have not been reported. Weining County has a humid subtropical monsoon climate, with an average of 1812 h of sunshine and 180 frostfree days per year, 926 mm of annual rainfall, small annual temperature differences and large daily temperature differences, and warm winters and cool summers, with an average temperature of 18 • in the summer [43,44]. The climatic conditions in Weining County are suitable for the occurrence of potato nematodes.
In Guizhou province, crop rotation is difficult to implement in many potato-producing areas; and chemical nematicides have problems of high toxicity, easy residues, and high costs, so biological control is a high priority. According to Mo [45], to solve some of the problems of nematode biological control, the competition for survival among and within groups of organisms in the soil ecosystem needs to be examined from the perspective of biodiversity. The fungal biological control of PCNs is an important component of integrated pest management for potatoes. However, very few fungal biological control agents are available on the market. This study aimed to investigate the strains and frequency of fungi colonizing cysts of G. rostochiensis collected from Weining County, Guizhou Province, China, and provide a basis for the control of G. rostochiensis.

Nematode Collection
The cysts of G. rostochiensis were collected from soils of one potato field naturally infected with G. rostochiensis, located in Weining County, Guizhou, China (Figure 1). In a field, ten 5 × 5 m grid plots were selected surrounding infected potato plants, and in each grid an approximate volume of 250 mL of soil was collected from the rhizosphere zone (0-20 cm in depth). The individual samples of each plot were collected and mixed in a bucket to obtain a single composite sample [46]. Each composite sample was thoroughly mixed to obtain a homogenous sample. A subsample of approximately 500 mL of soil was then air-dried at 37 • C for two days for PCN cyst extraction [47][48][49]. Cysts were extracted from a subsample of 50 g of dried soil using the Baunacke method [50,51], i.e., dried cysts that floated in water were decanted and collected on a 250 µM sieve.

Isolation of Fungi from Cysts of G. rostochiensis
The selected cysts were surface-sterilized in 2% H 2 O 2 for 3 min following three washes with distilled water. The surface-sterilized cysts were individually placed onto 1% WA plates. Plates were incubated at room temperature and monitored regularly. Fungal mycelia growing from the cultured cysts were re-cultured several times on new PDA plates. The pure cultures were initially grouped based on their morphological criteria. All fungal isolates were conserved in the Culture Collection of the Department of Plant Pathology, Agriculture College, Guizhou University.

DNA Extraction, PCR, and Sequencing
The fungal isolates were grown on PDA at 25 • C for 7 days. The resulting mycelia were then scraped off the surface of the plate with a sterile scalpel. Total genomic fungal DNA was extracted using a BIOMIGA Fungus Genomic DNA Extraction Kit (GD2416, BIOMIGA, San Diego, CA, USA) following the manufacturer's protocol. PCRs were conducted in a 25 µL reaction mixture containing 10 µL 2 × Bench Top Taq Master Mix (Biomiga, AT1201, San Diego), 7 µL of ddH 2 O, 1 µL of forward and reverse primers (10 µM/µL), and 1 µL of DNA template. PCR products were commercially sequenced with the same PCR primers used in the amplification reactions by Sangon Biotech Co., Ltd. (Shanghai, China).

Isolation of Fungi from Cysts of G. rostochiensis
The selected cysts were surface-sterilized in 2% H2O2 for 3 min following three washes with distilled water. The surface-sterilized cysts were individually placed onto 1% WA plates. Plates were incubated at room temperature and monitored regularly. Fungal mycelia growing from the cultured cysts were re-cultured several times on new PDA plates. The pure cultures were initially grouped based on their morphological criteria. All fungal isolates were conserved in the Culture Collection of the Department of Plant Pathology, Agriculture College, Guizhou University.

DNA Extraction, PCR, and Sequencing
The fungal isolates were grown on PDA at 25 °C for 7 days. The resulting mycelia were then scraped off the surface of the plate with a sterile scalpel. Total genomic fungal DNA was extracted using a BIOMIGA Fungus Genomic DNA Extraction Kit (GD2416, BIOMIGA, San Diego, CA, USA) following the manufacturer's protocol. PCRs were conducted in a 25 μL reaction mixture containing 10 μL 2 × Bench Top Taq Master Mix (Biomiga, AT1201, San Diego), 7 μL of ddH2O, 1 μL of forward and reverse primers (10 μM/μL), and 1 μL of DNA template. PCR products were commercially sequenced with the same PCR primers used in the amplification reactions by Sangon Biotech Co., Ltd. (Shanghai, China).

Multigene Analyses
Colonizing fungi were identified by protein-coding and ribosomal gene sequences. All forward and reverse sequences were used to create consensus sequences in BioEdit v. 7.0.9.0 [52], and BLASTn searches in NCBI were used to identify the taxonomic status at the genus level.

Multigene Analyses
Colonizing fungi were identified by protein-coding and ribosomal gene sequences. All forward and reverse sequences were used to create consensus sequences in BioEdit v. 7.0.9.0 [52], and BLASTn searches in NCBI were used to identify the taxonomic status at the genus level.

Dominant Taxa
A taxon is defined as dominant if Pi > Camargo's index (1/S), where S represents species richness, which is the number of fungal taxa, and Pi is calculated as the number of isolates (Ni) that belong to a certain taxon (i) divided by the total number of isolates (N) [53].

Lifestyle Diversity
The lifestyle status of culturable fungi was predicted using the FUNGuild database. The functional annotation of fungi at the genus level was considered appropriate [54].

In Vitro Parasitic Potential Tests of the Fungal Isolates towards Nematode Cysts
Fungi with different morphological characteristics were selected to screen potential parasitic fungi of G. rostochiensis cysts. The purified strains were cultured on a PDA medium. When the colony diameter grew to 1/2-3/4 of the culture dish, the surface-sterilized (2% H 2 O 2 for 3 min) cysts were individually placed onto the edge of the fresh hypha on the PDA medium, and 10 cysts were placed per dish, with three replicates per strain. After the cysts and plates were incubated at 25 • C for 10 days, the cysts were gently picked out (ensuring not to break them) and surface-sterilized in 0.5% NaClO for 3 min following three washes with distilled water. Each cyst was individually placed onto sterilized filter paper. After moisturizing the culture for five days, the cysts' colonization rate was recorded by observing the fungi growth on the cysts on the filter paper.

Data Analysis
All the statistical analyses were conducted in MS Excel and SPSS Statistics (version 19.0) software. Figures were generated using MS Excel, Adobe Photoshop 2021 and Chiplot Web (https://www.chiplot.online/, accessed on 26 December 2022).

Fungi Associated with Cysts of G. rostochiensis
Of the 200 examined cysts of G. rostochiensis, 80% were colonized by one to five or more different species of fungi, and 139 culturable strains were obtained by isolation and purification ( Table 1). The pure cultures were initially grouped based on their morphological criteria. The morphological characteristics selected for the observation were based on colonial color, mycelial shape, and growth rate. Among these strains, the colors were found to be: white, yellow, orange, red, gray pink, gray, purple, gray to brown, and brown to black. Various mycelial forms, such as compact, cottony, and airy, were observed. After seven days of cultivation, the mycelial growth rate of one strain (GUCC220042) was much lower than all other strains, only reaching 2 cm (diameter). Finally, forty-four fungal strains with different morphological characteristics were selected ( Figure 2). In total, 139 fungal strains were identified based on ITS sequence analyses and morphological observations. Forty-four isolates with different morphological characteristics were identified through multigene analysis of the combined internal transcribed spacer (ITS), 28S large subunit rDNA (LSU), and beta-tubulin (TUB). All sequences of the isolates were analyzed by NCBI-BLAST, representing 23 different genera.

Dominant Taxa
In all the isolates of the colonizing fungi, the Camargo index (1/S) at the order, family, and genus levels were 0.091, 0.059, and 0.043, respectively. Therefore, the dominant order was Hypocreales (62.6%); the dominant families were Nectriaceae (59.7%) and Aspergillaceae (12.9%); of the fungi identified, most were strains of Fusarium (58.9%) or Penicillium (10.8%). Fusarium was associated with 82 cysts, and Penicillium colonized 11% of the cysts. Absidia  (Figure 3). All the isolated fungi emerged from anywhere on the cyst surface.

Lifestyle Diversity
Twenty-three genera of colonizing fungi were analyzed for functional annotation in the FUNGuild database. No information was obtained for six genera-Arxotrichum, Aspergillus, Chaetomium, Paecilomyces, Phaeophlebiopsis, and Volutella. There were twelve different lifestyles represented by the remaining genera. Functional annotations of other genera are described in Figure 4. Plant pathogens and saprotrophs (wood, soil, and undefined saprotrophs) dominated the fungal communities, followed by endophytic fungi (4/23). Four genera, namely Fusarium, Mortierella, Trichoderma, and Xylaria, were found to have four or more lifestyles. Didymella and Fusarium are animal pathogens and can infect animals. Trichoderma are the only fungal parasites.

Lifestyle Diversity
Twenty-three genera of colonizing fungi were analyzed for functional annotation in the FUNGuild database. No information was obtained for six genera-Arxotrichum, Aspergillus, Chaetomium, Paecilomyces, Phaeophlebiopsis, and Volutella. There were twelve different lifestyles represented by the remaining genera. Functional annotations of other genera are described in Figure 4. Plant pathogens and saprotrophs (wood, soil, and undefined saprotrophs) dominated the fungal communities, followed by endophytic fungi (4/23). Four genera, namely Fusarium, Mortierella, Trichoderma, and Xylaria, were found to have four or more lifestyles. Didymella and Fusarium are animal pathogens and can infect animals. Trichoderma are the only fungal parasites.

Parasitic Potential of the Fungal Isolates Towards Nematode Cysts In Vitro
Forty-four fungal strains of varied morphology had different colonization rates on cysts. Most strains had a high colonization rate on cysts of G. rostochiensis (100%); the lowest was 16.7% (Table 2). Among the 44 fungi, the colonization rate of 40 fungal isolates to

Discussion
A total of 139 fungal strains were found on the cysts of G. rostochiensis from Weining County, Guizhou Province, China. The fungal isolates were assigned to three phyla, 11 orders, 17 families, and 23 genera. These fungi belonged to the genera Fusarium, Penicillium, Absidia, Arxotrichum, Aspergillus, Chaetomium, Clonostachys, Coriolopsis, Crinipellis, Didymella, Edenia, Gongronella, Mortierella, Nigrospora, Paecilomyces, Paraphaeosphaeria, Peroneutypa, Pestalotiopsis, Phaeophlebiopsis, Phanerochaete, Trichoderma, Volutella, and Xylaria. Forty-four strains with different morphological characteristics were selected from 139 fungi, which all had a certain colonization rate to cysts in vitro. In 44 strains, 27 demonstrated a colonization rate of 100% on the cysts of G. rostochiensis. Colonized fungi were isolated from G. rostochiensis for the first time in China, and the taxonomic diversity of the fungi from G. rostochiensis were preliminarily clarified.
In previous studies, there have been many reports on colonizing fungi associated with cyst nematodes, most of them concerning Heterodera spp., with only a few on Globodera spp. In Siwi's study, out of 123 fungal isolates obtained from PCN cysts and PCN-infested soils in Indonesia, 12 isolates showed egg-and cyst-parasitic abilities of over approximately 50%, which were identified as Gliocladium virens, F. oxysporum, F. lateritium, P. tritinum, P. oxalicum, and Taralomyces spp. [55]. The fungi isolated in this study were mostly opportunistic fungi, a class of fungi that specifically or facultatively colonize the cysts of plant-parasitic nematodes, including a large number of soil-dwelling fungi [56]. Fusarium, Paecilomyces, and Mortierella, the most commonly isolated genera, were also isolated in this study [57]. Combining the results of previous studies, we found that the dominant genera on the cysts of PCNs were Fusarium and Penicillium. Fusarium was the most abundant genus. Although the isolation rate of Fusarium on cysts is very high, whether Fusarium is pathogenic to potatoes and whether it is suitable as a common plant-pathogenic fungus for biocontrol fungi remains to be further studied.
The percentages of cysts, eggs, and females of cyst nematodes colonized by fungi in agricultural soil ranged from 10 to 90%, with about 50% being the most common species [84,85]. In this study, we obtained 40 isolates showing cyst-colonizing abilities of over 50%. Currently, two possible routes for the biological management of plant-parasitic nematodes have been proposed. One is to mass produce an effective nematode-destroying fungus in the laboratory and then apply it to soils [86]. The other is to enhance the natural nematophagous fungal populations in soils by altering their surrounding conditions. However, the commercial success of these approaches has been limited; nevertheless, there are encouraging reports concerning reducing nematode populations by adding certain kinds of amendments, such as chitin and green manure crops, to soils [87,88].
Although not considered as traditional biological control, another promising approach by which nematophagous fungi, as well as other soil fungi, can be used for developing new means to control animal-and plant-parasitic nematodes is to use antagonists as a source for isolating new compounds with nematicidal activity [89]. Our study focuses on the diversity and parasitic potential of colonizing fungi isolated from G. rostochiensis in order to better understand their ecology. For the development and stability of nematode biocontrol agents, it is critical to analyze the biocontrol potential of these colonizing fungi and the soil ecology of the colonized microorganisms.