Chemical Signals of Vector Beetle Facilitate the Prevalence of a Native Fungus and the Invasive Pinewood Nematode

Identification and diversity of ophiostomatoid fungi: A total of 116 pupal chambers of M. alternatus from Pinus massoniana trees were collected from four different locations belonging to three districts of Guangdong province (Table 1). Sampling was done from five wilted pine trees (killed because of pine wilt disease within a year) selected at random at least 20 m apart from each other.

To isolate and identify fungus, wood tissues from pupal chambers were chopped and placed directly onto prepared fungal growth medium (20 g Biolab malt extract, 20 g Biolab agar, and 1 liter deionized water, amended with 0.05% cycloheximide and 0.04% streptomycin). The cultures isolated in this study were archived in the culture collection of the Forestry and Agricultural Biotechnology Institute, University of Pretoria, Pretoria, South Africa.

Pure cultures of isolated fungi were sorted into groups based on their morphology. The anamorph structures originated from asexual reproduction were mounted in lactic acid on glass slides and examined microscopically. To induce the production of the fungal fruiting body, perithecia, anamorphic isolates were cultured on 1.5% oatmeal agar (15 g oat powder, 20 g Biolab agar, and 1 liter deionized water) and 2% wateragar (WA) medium (20 g Biolab agar and 1 liter distilled water) with sterilized pine twigs at 25°C in the dark.

To extract and sequence DNA, single hyphal tip cultures were prepared. Each culture was grown in 50 ml malt extract broth (30 g Biolab malt extract and 1 liter distilled water) at 25°C in the dark for 10 d. Mycelium was then harvested and freeze-dried after filtration through Whatman no. 1 filter paper and was ground into a fine powder. DNA was extracted from the resulting powder using PrepMan Ultra Sample reagent (Applied Biosystems, Foster City, CA) following the manufacturer’s protocols.

PCR and DNA sequencing reactions were prepared and performed as previous works (Lu et al., 2009). Data sets were compiled in MEGA 5 (Tamura et al., 2007) and the sequences were deposited in GenBank under the accession numbers provided in Table 2. National Center for Biotechnology Information similarity scores were obtained using a BLAST search of sequences previously deposited in GenBank (www.ncbi.nlm.nih.gov).

The species diversity of fungal isolates from different sites was compared. The number of species (S), Simpson’s index (D = Sum [Pi²], where Pi is the number of a given species divided by the total number of isolates observed) and Simpson’s index of diversity (1 – D) were used to evaluate species diversity (Lu et al., 2009).

Interaction of associated ophiostomatoid fungi: To determine the effects of a fungus on another related species, three fungal species, i.e., Sporothrix sp. 1, Sporothrix sp. 2, and O. ips were tested through a modified pairwise cultivation method (Hofstetter et al., 2005). The fungal isolates from both the Zhejiang and Guangdong provinces were used in these experiments. Specifically, a mycelial disc (5 mm diameter) was taken out from the periphery of an actively growing 10-d-old culture on 2% malt extract agar (MEA: 20 g malt extract, 20 g agar, and 1 liter deionized water) and was placed on a 90 × 20 mm petri dish containing 20 ml of 2% MEA. The two different inoculates were placed on opposed edges of the petri dishes and incubated at 25°C in the dark. Ten replicates for each treatment, including the controls (the same species of fungi on two sides of the plate), were performed. Radial growth for each species was measured along the line (ca. 5 cm in length) between two inoculates after 6 d, and the proportion of each species accounting for the line was calculated. Arcsine transformation was performed, where required to normalize the data, and paired t test was used to test the significance of difference. All statistical tests were conducted using the SPSS Statistics v. 19.0 (SPSS, Inc., Chicago, IL).

Effects of FAEE on mycelial growth: To determine the effects of FAEE on the fungal growth, Sporothrix sp. 1, Sporothrix sp. 2, and O. ips were cultured on the medium as described earlier. The most common FAEE compounds, i.e., EP, EL, ES, and EO were used as test treatments at the concentrations of 32.37, 42.55, 30.33, and 104.28 μg/ml, respectively. These concentrations were consistent with those, the nematodes would experience in a beetle chamber containing a single beetle (Zhao et al., 2013b). For each treatment, 1 ml of each test solution was added to a sterile filter paper (80 mm diameter) that was placed inside the lid of a petri dish and sealed with parafilm (Bemis Company, Neenah, WI). The treated plates were incubated at 25°C in the dark. Ten petri dish cultures for each treatment including two controls (filter paper with 1 ml ethanol and filter paper only) were used. Radial growth was measured in four directions (0°, 90°, 180°, and 270°) from the center of cultures every 24 hr for Sporothrix sp. 1 and 48 hr for the other two fungal species until the fungi reached the edges of the petri dish. The experiments were repeated with fungal cultures isolated from Zhejiang province. The growth rates were used for statistical analysis. One way analysis of variance (ANOVA) was performed for each fungal species, respectively.

Effects of fungi on the fitness of PWN: Five pairs of adult nematodes were inoculated to each of the three fungal species cultivated on 2% MEA (10 plates per fungus) and were incubated at 25°C in the dark for 9 d. After incubation, live nematodes were recovered using Baermann funnel. The number of individual nematodes was counted, and the body length of female, male, and the J_III of PWN (n ≥ 50 nematodes from each fungal species) were measured using an Olympus BX51 microscope with a digital imaging system Olympus DP2-BSW software (Niu et al., 2012). One-way ANOVA was performed for statistical analysis. To confirm the consistency and repeatability of the results, all the experiments were repeated with the fungal species collected from Zhejiang province.

Species diversity of ophiostomatoid fungi: Among all surveyed sites, we found a significant number of pupal chambers in recently killed trees at all of the four surveyed sites. Moreover, all surveyed beetle chamber was infested with PWN (Table 1).

In total, 65 ophiostomatoid fungal isolates were recovered from M. alternatus chambers during the course of this study from Guangdong province (Table 3). Small numbers of other fungi including yeasts were isolated, but these were not included in this study.

Based on culture and anamorph morphology, isolates could be divided into three groups. Two groups presented Sporothrix anamorph in the cultures, whereas the remaining groups had a Pesotum-like anamorph. Comparing with the referenced sequences in GenBank, two new Sporothrix species and O. ips were identified (Table 2).

More isolates were retrieved from Longtoushang (number of species: S = 18 and Simpson’s index of diversity: 1 – D = 0.66) and Datianshan (number of species: S = 17 and Simpson’s index of diversity: 1 – D = 0.65), both of which belong to Huangpu district, than those from Baiyunshan (number of species: S = 15 and Simpson’s index of diversity: 1 – D = 0.66) and Conghua district (number of species: S = 15 and Simpson’s index of diversity: 1 – D = 0.66). For each fungus, the number of isolates ranged from four to seven in different collection sites. However, we sampled more of the two newly found species in China, i.e., Sporothrix sp. 1 (number of species: S = 24 and frequency = 36.9) and Sporothrix sp. 2 (number of species: S = 23 and frequency = 35.4), compared with the common O. ips (number of species: S = 18 and frequency = 27.7) (Table 3).

Interaction of associated ophiostomatoid fungi: When cultured together, the growth of Sporothrix sp. 1 was significantly faster than that of O. ips (Guangdong: t = 9.23, P < 0.001 and Zhejiang: t = 10.45, P < 0.001) and Sporothrix sp. 2 (Guangdong: t = 42.13, P < 0.001 and Zhejiang: t = 48.46, P < 0.001) (Fig. 1). At the same time, O. ips grows significantly faster than Sporothrix sp. 2 (Guangdong: t = 20.55, P < 0.001 and Zhejiang: t = 23.46, P < 0.001) (Fig. 1), whereas the two inoculates in all controls (paired cultured with the same species) have no difference (P > 0.05 in both Guangdong and Zhejiang) (Fig. 1).

FIG. 1.

The effect of beetle surface compounds on the fungal growth: The growth bioassays of different fungi using the four main FAEE from the beetle surface showed that EP and EL significantly improved the growth of Sporothrix sp. 1 (Fig. 2A,B), but had no effects on the other two species of fungi (Fig. S1). The other two FAEE (EO and ES), however, did not exhibit any activity on the growth of all of the three fungi (Figs. 2,S1).

FIG. 2.

The effects of fungi on the fitness of PWN: For the organisms from Guangdong, the fecundity of nematode and their body length were both significantly affected by the fungal species they were cultured on. The number of nematodes reared on Sporothrix sp. 1 was highest, then O. ips, and Sporothrix sp. 2 (one-way ANOVA: F = 33.31, P < 0.001; Fig. 3A). The highest body length of female (one-way ANOVA: F =84.05, P < 0.001; Fig. 3B), male (one-way ANOVA: F = 35.11, P < 0.001; Fig. 3C), and J_III (one-way ANOVA: F = 89.03, P < 0.001; Fig. 3D) were recorded in the nematodes cultured on Sporothrix sp. 1, followed by those reared on O. ips, whereas significantly smaller lengths of each stage were recorded when they were reared on Sporothrix sp. 2. The experiments using the fungi and nematodes from Zhejiang showed the consistent results, except that the body length of J_III have no difference when fed on either Sporothrix sp. 1 or O. ips (Fig. S2).

FIG. 3.

The coexistence of PWN, its vector beetle, and the associated blue stain fungus in diseased trees represents their symbiotic association (Fig. 4) In this association, the influences of the beetles or ophistomatoid fungi on PWN and ophistomatoid fungi on vector beetle have been intensively reported (Linit, 1988; Necibi and Linit, 1998; Yang et al., 2003; Sun, 2005; Akbulut and Stamps, 2012; Zhao et al., 2013a, 2016). However, the effect of vector beetle on the fungi has not been reported. Here, we have elucidated the underlying chemical basis of the symbiotic association between vector beetle and Sporothrix sp. 1. Our findings demonstrate that FAEE (EP and EL) produced on beetles’ body surface enhance the growth of Sporothrix sp. 1 in a way that the fungus grows faster than its competitive species, O. ips and Sporothrix sp. 2. This ultimately lead to higher numbers of PWN that develop on Sporothrix sp. 1, with increased population number and body size of both adults and the dispersal juveniles (J_III). These findings complement to earlier reports stating that nematodes fed on Sporothrix sp. 1 have a better reproductive ability because of the DAA produced by this fungal species (Zhao et al., 2013a). Our results suggest that Sporothrix sp. 1 gets direct chemical signals from the beetles to expand and exploit a greater niche, ultimately escalating the pathogenicity of nematodes.

FIG. 4.

Furthermore, isolation of Sporothrix sp. 1 from all the sampled pupal chambers depicts a strong association of the fungi with vector beetles. FAEE (C16 and C18) from Monochamus beetle are known as chemical signals synchronizing the beetle to PWN life cycle (Zhao et al., 2013b, 2016). We also found the extended scope of these FAEE, especially the EP and EL, which enhanced the growth of Sporothrix sp. 1 (Fig. 4). This symbiosis might provide a new aspect of explanation to invasive success of PWN in China.

The successful establishment of the invasive pathogen in the introduced region is often attributed to local associates that enhance their pathogenicity. In China, PWN might have developed a symbiotic association with two of the native fungal species (Sporothrix sp. 1 and Sporothrix sp. 2), which are absent in North America, the native region of B. xylophilus. When developed on Sporothrix sp. 1, nematodes increase in body sizes and had higher fecundities, hence, favoring pine wilt disease prevalence in china. These findings are also in accordance with the earlier studies reporting that larger body size in worms produce more offspring (Poulin, 1996).

Besides nematodes, cerambycid beetles are also known to establish mutualistic associations with other microorganisms including mites (Whitney, 1982; Paine et al., 1997; Moser et al., 2005; Hofstetter et al., 2006; Cardoza et al., 2008) that might be responsible for transferring fungi from one cerambycid pupal chamber to the other (Wingfield, 1987). But few studies focus on the role of mites in the cerambycid beetles—nematodes system. Therefore, to complete the picture of pine wilt disease system, future research is necessary to establish the role of mites in the transmission of fungi.

In conclusion, the results present a unique aspect in the symbiotic relationship among vector beetle, associated blue stain fungi, and the PWN. The vector beetle, M. alternatus, not only directly disseminates PWN to new hosts but also improves the quality of fungal diet of PWN, hence, indirectly facilitating its prevalence. This information will also contribute to a better understanding of the microecological interactions that facilitate the eruptive population dynamics of important forest pests (Hofstetter et al., 2006).