The pine sawyer beetle M. alternatus was collected using commercial traps (FEILUOMENG Co., China) baited with attractants in naturally infested pine forests from five geographical regions of southern China ( Figure S1 ). The specimens were labeled and transported to the laboratory in individual sterilized tubes for each beetle with fresh twigs. During rearing of the collected beetles (approximately 500 beetles in total), 81 beetle cadavers or symptomatically living ones with fungal infections were observed and stored at 4°C for use in this study.

Fungal isolation was performed under aseptic conditions following previously described procedures (Parker et al., 2003). The beetle body was divided into seven positions using sterilized scissors: antennae, head, thorax, abdomen, eggs (for females), wings, and legs. Tissues were inoculated onto potato dextrose agar (PDA) plates containing 0.05 g/L streptomycin, penicillin G and tetracycline and then cultured in an incubator at 25°C until the tissues were covered and surrounded by mycelia. Fungal purification was repeated twice on PDA with antibiotics, and pure isolates were cultured on conventional PDA and deposited in the Key Laboratory of Vector Biology and Pathogen Control of Zhejiang Province at Huzhou University.

The fungal isolate was cultured in 50 ml sterile potato dextrose broth (PDB) at 25°C with shaking at 180 rpm in a 250ml flask for 5–7 days. Mycelia harvested from the PDB were separated by filtration and homogenized in liquid nitrogen using a pre-cooled mortar and pestle. Genomic DNA was extracted using the Rapid Fungi Genomic DNA Isolation Kit (Sangon Biotech Co., China), and the rDNA-ITS region was amplified using primer pairs ITS1 and ITS4 (White et al., 1990) via the following procedure: initial denaturing at 95°C for 4 min, followed by 35 cycles of denaturing at 94°C for 60 s, annealing at 58°C for 60 s, elongation at 72°C for 2 min, and a final elongation at 72°C for 10 min. Amplified PCR products were sequenced and compared with the ITS sequence database in GenBank using BLAST software on the National Center for Biotechnology Information (NCBI) website. The gene sequences were deposited in NCBI under GenBank accession numbers OP321299–OP321543.

Based on the above results, eleven representative fungal species, namely, Aspergillus austwickii, A. ruber, Beauveria bassiana, Clonostachys rosea, Lecanicillium attenuatum, L. aphanocladii, Penicillium citrinum, Pestalotiopsis disseminata, Purpureocillium lilacinum, Scopulariopsis alboflavescens, and Trichoderma dorotheae, were used to test their entomopathogenic activities. Considering that it is difficult to collect an adequate quantity of adult M. alternatus with long-lasting alive status from the field to match this bioassay, the population of the model insect beetle Tribolium castaneum, successfully reared in the laboratory with a common genetic background, was used to determine the entomopathogenicity of the eleven fungal isolates. Their infection phenotypes on M. alternatus were also further confirmed in the subsequent experiments. Conidia obtained from 14-day-old fungi were suspended in a sterile aqueous solution of 0.01% Tween-80 (Orduño-Cruz et al., 2015). This suspension was shaken with glass beads and filtered through two layers of gauze to remove the mycelia. The conidia were counted with a hemocytometer and adjusted to 1×10⁸ conidial/ml with sterile 0.01% Tween-80.

Adult T. castaneum was reared on wheat bran at 25°C in a climate-controlled incubator. Beetles were surface-sterilized with bleach, ethanol, and distilled water [10:10:80 (vol:vol)] and starved for 24 h before use. Aliquots of wheat bran were UV-sterilized, placed in sterile Petri dishes, and then treated with conidial suspension. T. castaneum beetles were also immersed in the conidial suspension for 10 s and transferred to a Petri dish (n = 20 in each Petri dish). The wheat bran was replaced with a new conidial treatment every 4 days. Beetles and wheat bran in the control group were treated with sterile 0.01% Tween-80 solution. Each fungal and control group was replicated three times. In a pilot assay, beetles were checked for mortality 9 days after fungal treatments and in the subsequent survival monitoring assay, beetles were recorded daily for 15 days. Dead beetles from these assays were transferred to sterile moist centrifugal tubes and inspected for fungal growth on the cadavers. The aerial hyphae of each fungal species on cadavers were confirmed using Koch’s postulate.

Fungal entomopathogenic activity correlated well with the activities of three enzymes including protease, chitinase, and lipase, in M. alternatus and many other insect pests (Cortez-Madrigal et al., 2014; Dhawan and Joshi, 2017; Moharram et al., 2021). The enzyme activities of the fungal species in this study were measured following previously reported procedures (Sharma et al., 2016; Li et al., 2018; Alabdalall et al., 2020) with minor modifications. To test protease activity, conidia (1×10⁸ conidia/ml) of each isolated fungus were cultured in 50 ml of protease-inducing medium with shaking at 180 rpm at 25°C for 10 days. One milliliter of fermentation supernatant was obtained from each culture solution every 2 days and incubated with 1 ml of 1% casein solution at 37°C for 30 min. The quantities of the reaction products were determined by measuring the optical density at 600 nm (OD600) using the Folin-phenol reagent method. The negative control of each sample was prepared by adding trichloroacetic acid to the reaction solution before incubation to inhibit putative enzymatic activities. Five concentrations of tyrosine were used to create a standard curve, and one protease unit was defined as the amount of fermentation supernatant required to produce 1 µg of tyrosine from casein per minute. To test chitinase activity, conidia (1×10⁸ conidia/ml) of fungi were cultured in liquid chitin medium for 10 days and at every 2 d-interval, 1 ml of fermentation supernatant was incubated with 1 ml of 1% colloidal chitin at 50°C for 1 h. The negative control for each sample was prepared by heating the reaction solution in a boiling water bath before incubation. Reaction product quantities were determined by measuring the OD540 using the 3,5-dinitrosalicylic acid (DNS) method. Five concentrations of N-acetyl-D-glucosamine were used to create a standard curve, and one chitinase unit was defined as the amount of fermentation supernatant required to produce 1 µg of N-acetyl-D-glucosamine per minute. To test lipase activity, conidia (1×10⁸ conidia/ml) of fungi were cultured in Sabouraud’s dextrose agar (SDA) medium for 10 days and 0.2 ml of fermentation supernatant was pipetted every 2 days and incubated with 0.2 ml matrix solution containing 4-nitrophenyl palmitate (Gopinath et al., 2005) at 37°C for 30 min. The negative control for each sample was prepared by adding trichloroacetic acid to the reaction solution before incubation. The quantity of the reaction product p-nitrophenol was calculated by comparing the OD410 value with the linear standard curve, and one lipase unit was defined as the amount of fermentation supernatant required to produce 1 µg of p-nitrophenol per minute. Three biological replicates were used for enzymatic bioassays.

The fungal species with remarkable entomopathogenic activities were cultured on PDA plates for 7–11 days at 25°C. Circular agar blocks (5 mm in diameter) from colonies were transferred to new PDA plates to observe colony morphology. For asexual morphological descriptions, conidia, conidiophores and nutritional hyphae were sampled from colonies on glass slides, and their traits were observed and measured using a Leica DM2000 microscope (Leica Co., Germany).

Fungal infection phenotypes in live M. alternatus adults (sampled from Zhejiang Province) were confirmed following the procedure described above. Beetles were surface-sterilized, dipped in conidia suspension, transferred to 50-ml sterile tubes (one beetle per tube), and provided with fresh pine twigs (also immersed in conidia suspension for 10 s). Beetle activity was assessed based on the quantity of the frass produced. When feeding ceased, the beetle was transferred to a new 50-ml tube (with sterile moist cotton) for fungal growth for 7 days. Each fungal species was applied to five living beetles. The infected beetles were observed as described above and fulfilled by Koch’s postulates.

For scanning electron microscopy (SEM) observation, colony plugs and infected beetles were fixed in pre-chilled 2.5% glutaraldehyde at 4°C for 2 days. Samples were washed thrice in 0.1% phosphate buffer (pH 7.2–7.4) for 5 min and dehydrated in 30%, 50%, 70%, 80%, 90%, 95%, 100% ethanol for 10 min. The samples were dried in a vacuum freeze dryer (Yamato Scientific Co., Japan), coated with platinum using a sputter coater, and observed using an S-3400N scanning electron microscopy (Hitachi, Japan).

The primer pairs NS1/NS4 (White et al., 1990), LR7/LROR (Vilgalys and Hester, 1990; Rehner and Samuels, 1994), EF-983F/EF-2218R (Aphidech and Kusavadee, 2013), RPB2-5’F/RPB2-5’R (Wang et al., 2015), and TUB1/TUB22 (Qi et al., 2021), were used to amplify a region spanning of the nuclear ribosomal SSU gene, a segment of the large subunit rRNA gene (LSU), part of the elongation factor 1-alpha (EF-1α) gene, the second largest subunit sequences of RNA polymerase ІІ (rpb2), and part of the β-tubulin gene, respectively. The PCR products were examined by 1.5% agarose gel electrophoresis and then subjected to sequencing with the GenBank accession numbers listed in Table S1 . Sequences of ITS and the five genes were then aligned using Clustal X2.0 and MEGA 6 (Larkin et al., 2007; Tamura et al., 2013). Ambiguously aligned sites were excluded, and gaps were treated as missing data during sequence alignment. The aligned sequences of the six genes were concatenated to construct Maximum Likelihood (ML) phylogenetic trees using MEGA 6.

Two- to three-year-old P. massoniana seedlings were used to determine the phytopathogenic activities of the entomopathogenic fungi. Fungal inoculation was conducted by making a wound on the main stem of each P. massoniana seedling using a sterile cork borer with a 5-mm diameter at 15 cm above the soil line (one seedling with one inoculation point). A plug with a 5-mm diameter was taken from the margin of one actively growing fungal species cultured on PDA and transferred to the cambium layer of seedling inoculation point. The inoculation points were wrapped in a laboratory film (Parafilm M, USA) to prevent contamination and desiccation. Mock inoculation, mimicked using a plug of PDA alone (without fungi), was applied to the seedlings in the same manner as a control. The Fusarium species, which are broad-spectral plant pathogens, together with other non-entomopathogenic fungi were also included in the experiment. Each fungal isolate and the control were replicated thrice. After 3 weeks, lesion length was measured both downward and upward from the inoculation point. The fungi were re-isolated from the infected areas to complete Koch’s postulates.

To measure pectinase and cellulase activities, conidia (1×10⁸ conidia/ml) of each fungus were cultured in 50 ml of pectinase- or cellulase-inducing medium (0.2% KNO₃, 0.05% KCl, 0.001% FeSO₄, 0.1% K₂HPO₄, 0.05% MgSO₄·7H₂O, 1% pectin, or 1% carboxymethyl cellulose sodium; pH 5.0) for 10 days. For every 2 days, 1 ml of fermentation supernatant was pipetted and incubated with 1 ml of 0.1% pectin solution or 0.5% sodium carboxymethyl cellulose solution at 50°C for 1 h. The negative control for each sample was prepared by heating the reaction solution in a boiling water bath for 5 min before incubation. After incubation, the reaction solutions were bathed in boiling water, incubated with 2 ml of DNS, boiled again at 100°C for 5 min, and finally refrigerated on ice. Reaction product quantities were determined by measuring the OD540 using the 3,5-dinitrosalicylic acid (DNS) method (Pereira et al., 2002). Five concentrations of D-galacturonic acid and D-glucose were used to generate the standard curves. One pectinase unit and one cellulase unit were defined as the amounts of fermentation supernatant to produce 1 µg of D-galacturonic acid and 1 µg of D-glucose per minute, respectively. The enzymatic bioassays were performed in triplicate.

Variations in α-diversity indices of fungal communities among geographical regions, mortality of beetles caused by fungi at 9 d, enzymatic activity levels, and phytopathogenicity among fungi were assessed using one-way ANOVA followed by the Bonferroni approach for pair-wise comparisons. Where normality and/or equal variance were not assumed, nonparametric Kruskal-Wallis one-way ANOVAs were performed, followed by pair-wise comparisons using the Mann-Whitney U test. Absolute abundance was estimated as the number of isolates per fungal taxa, whereas the ratio of isolates from each fungal taxa to total fungal isolates was considered the relative abundance of certain taxa. Venn diagrams and upset plots were used to display the intersection of fungal species in beetle geographical populations (or body positions or genders) using R software with the package ggplots2. Principal component analysis (PCA) was used to investigate the variation patterns of the fungal community structures among geographical locations. Sörensen’s similarity index (Cs) was calculated using the equation: ( Cs = 2 c a + b ) (Sörensen, 1948), where a and b are the numbers of species unique to each geographical location and c is the number of shared species between the two locations. Principal coordinate analysis (PCoA) was applied to visualize the relationship between the variation in fungal community composition and beetle geographical populations (or body positions or genders), followed by significance tests using one-way PERMANOVA. The survival of beetles was calculated using Kaplan-Meier survival analysis. Comparisons between survival curves were further tested using the Log Rank (Mantel-Cox) method. GraphPad Prism 6 and PAST software were used for statistical analyses.

A total of 640 fungal strains were isolated from 81 beetle cadavers or those symptomatically alive in five geographical regions, belonging to 15 fungal genera and 39 species ( Table 1 ). Genus Aspergillus was highest in relative abundance (35.47%), followed by Penicillium (25.31%), Scopulariopsis (9.69%), Lecanicillium (8.75%), Fusarium (7.66%), and Trichoderma (6.42%). These genera accounted for 93.30% of the total identified strains. The most dominant species was A. ruber (21.72%), followed by P. citrinum (9.84%), A. sydowii (9.84%), S. alboflavescens (9.69%), L. attenuatum (8.44%), and F. annulatum (5.16%). These collectively represented 64.69% of the total identified strains.

Significant differences in fungal community composition were found among geographical regions/provinces in which M. alternatus adults were sampled. In Zhejiang, a total of 197 fungal strains were isolated from the beetles, belonging to 11 genera and 22 species, in which fungal species L. attenuatum and P. citrinum were dominant, with relative abundances of 25.38% and 14.72%, respectively ( Table S2 ; Figure S2 ). In Sichuan, a total of 36 fungal strains were isolated, belonging to 6 genera and 12 species, in which fungal species A. austwickii and T. dorotheae were dominant, with relative abundances of 41.67% and 19.44%, respectively ( Table S2 ; Figure S2 ). In Fujian, 172 fungal strains belonging to 12 species and 6 genera were isolated, in which fungal species S. alboflavescens and A. sydowii were higher in relative abundances than others, at 31.40% and 29.07%, respectively ( Table S2 ; Figure S2 ). In Guangdong, 74 fungal strains belonging to 14 species and 9 genera were isolated, and P. citrinum with a relative abundance of 22.97%, was higher than other species ( Table S2 ; Figure S2 ). In Guangxi, 161 fungal strains belonging to 6 species and 5 genera were isolated, of which A. ruber with a relative abundance of 85.09% was the dominant species ( Table S2 ; Figure S2 ). The fungal species, namely, L. attenuatum, A. austwickii, S. alboflavescens, and A. ruber, appeared to be region-representative in Zhejiang, Sichuan, Fujian, and Guangxi, respectively, since their relative abundances were highest in one population while strikingly low in the others. Since P. citrinum was also abundant in Zhejiang, no region-representative species were found in Guangdong.

Additionally, except for the fungi isolated from M. alternatus in Fujian population, fungal communities from the rest of geographical populations contained distinct species (exclusively isolated species) in their respective populations ( Figure 1A ). Furthermore, the number of shared fungal species in the Fujian and Guangdong population was higher than that in the other populations ( Figure 1A ).

The α-diversity index comparisons of fungal communities among populations and multivariate analyses further supported the geographical variation in community composition. An obvious decrease in fungal species diversity was found in high- to low-latitudinal populations ( Table S3 ). Species richness and diversity indices were significantly higher in the population of Zhejiang than those of Guangxi, whereas dominance and evenness indices were higher in the Guangxi ( Table S3 ). Principal component analysis (PCA) showed that fungal community compositions of M. alternatus in Zhejiang, Fujian, and Guangxi populations diverged remarkably from each other ( Figure 1B ), and the pair-wise similarity coefficient calculation further indicated that fungal communities from the three regions were dissimilar to each other, while the fungal composition of the Fujian population was more similar to the Guangdong than other populations ( Table S4 ). Principal coordinate analysis (PCoA) elucidated the distances of fungal community composition between geographical population groups and inter-sample variations within groups ( Figure 1C ; one-way PERMANOVA; F = 9.180, P = 0.0001). Pair-wise comparisons also demonstrated strikingly significant mutual dissimilarities in fungal composition between different M. alternatus geographical populations, except for those isolated from Fujian and Sichuan populations, which did not show notable differences with that from Guangdong population ( Table S5 ).

Fungal communities from the five geographical populations were integrated and re-categorized according to body position ( Table S6 ; Figures S3A, B ) and gender ( Table S7 ; Figures S4A, B ). However, no significant differences were found in the fungal community composition among M. alternatus body positions (one-way PERMANOVA; F = 1.135, P = 0.217) and among genders (one-way PERMANOVA; F = 1.167, P = 0.277), as visualized by PCoA analyses ( Figures S3C, S4C ).

Fungal isolate Beauveria bassiana, a well-known entomopathogenic fungus, as well as four more fungal isolates with activities reported previously in insects including Clonostachys rosea (Mohammed et al., 2021), L. aphanocladii (Nedveckytė et al., 2021), Pestalotiopsis disseminata (Lv et al., 2011) and Purpureocillium lilacinum (Panyasiri et al., 2022), together with A. austwickii, A. ruber, L. attenuatum, P. citrinum, S. alboflavescens and T. dorotheae—which were the main species associated with their corresponding M. alternatus populations—were used to evaluate their entomopathogenic activities. In a pilot assay, after 9 days of infection, four fungal species, namely, A. austwickii, B. bassiana, L. attenuatum, and S. alboflavescens showed significantly higher mortality on the model insect beetle T. castaneum than the control ( Figure S5 ; Kruskal-Wallis test; χ₁₀ ² = 25.86, P = 0.0039). In the subsequent 15-day-time course assay, there was a significant difference in the survival curves among treatments ( Figure 2A ; Kaplan-Meier test; χ₁₁ ² = 288.9, P = 0.0001). No significant differences were found between the survival of T. castaneum adults inoculated with Tween 80 (control group) and those inoculated with conidial suspension of P. lilacinum, P. disseminata, C. rosea, and L. aphanocladii ( Figure 2A ; Log-rank tests; Pl, χ₁ ² = 0.22, P = 0.6398; Pd, χ₁ ² = 0.44, P = 0.5080; Cr, χ₁ ² = 0.57, P = 0.4511; Lap, χ₁ ² = 1.35, P = 0.2456), indicating that the four fungal species did not influence beetle fitness. However, the survival of T. castaneum adults inoculated with B. bassiana, A. austwickii, S. alboflavescens, T. dorotheae, A. ruber, and L. attenuatum was significantly lower than that of the beetles in the control group ( Figure 2A ; Log-rank tests; Bb, χ₁ ² = 96.23, P< 0.0001; Aa, χ₁ ² = 101.6, P< 0.0001; Sa, χ₁ ² = 41.75, P< 0.0001; Td, χ₁ ² = 29.89, P< 0.0001; Ar, χ₁ ² = 26.11, P< 0.0001; Lat, χ₁ ² = 20.78, P< 0.0001), demonstrating the potent insecticidal activities of the six fungal species. Another fungus, P. citrinum, showed a relatively moderate efficacy in killing T. castaneum adults ( Figure 2A ; Log-rank test; χ₁ ² = 7.70, P = 0.0055).

The entomopathogenic activities of these fungal species were determined using enzymatic activity assays. A. austwickii and B. bassiana had consistently higher protease activities with incubation time than the other fungal species ( Figure 2B ). B. bassiana performed better in chitinase activity than A. austwickii, reaching a peak level higher than that of other fungi on day 8 ( Figure 2C ). Except for B. bassiana, the other fungi showed decreased chitinase activity from day 4 until the end, and S. alboflavescens exhibited a relatively higher chitinase activity during this time ( Figure 2C ). As for lipase activity, A. austwickii reached a peak level higher than other fungi on day 2 and then exhibited a decrease in activity to a stable level ( Figure 2D ). Both B. bassiana and L. attenuatum showed the highest lipase activity levels on day 6, compared to other fungi ( Figure 2D ). P. citrinum did not exhibit an increase in lipase activity until day 8–10 of the incubation ( Figure 2D ).

Among the seven entomopathogenic fungi that caused significant fitness loss in T. castaneum adults, three fungi (A. ruber, P. citrinum, and T. dorotheae; grown on PDA medium; Figures S6–S8 ) did not display visible infection phenotypes on the beetle, neither did they on M. alternatus. However, the other four (A. austwickii, B. bassiana, L. attenuatum, and S. alboflavescens) showed conspicuous infection symptoms on T. castaneum body surface ( Figure S9 ), indicating strong parasitic capacities of these fungi. Their infection phenotypes were also observed in M. alternatus adult bodies, with mycelia of the four fungal species penetrating the body surface from the inside of the beetle, carrying asexual conidiophores ( Figures 3 – 6 ).

The asexual morphological features of parasitic entomopathogenic fungi grown on PDA medium and the bodies of the two beetle species were observed and as follows: A. austwickii colony was initially white, and then became dark green due to the production of spores ( Figure 3A ). The reverse color was brown and green ( Figure 3B ). The conidiophores were smooth-walled and contained pyriform-shaped vesicles ( Figure 3C , by optical microscope, OM). The conidia were globose, 3.5–4.4 μm×3.1–3.6 μm in diameter ( Figure 3D , by OM) and grown from ampulliform phialides on the conidiophore ( Figure 3E , by SEM). The conidia were shown with rough walls ( Figure 3F , by SEM). M. alternatus cadaver was covered with yellowish green mycelia of A. austwickii ( Figure 3G ; mainly on its abdomen). Conidiophores grown from M. alternatus ( Figure 3H , by SEM) and T. castaneum beetles ( Figure S9A ) had radial conidial heads typical of the Genus Aspergillus, producing spherical conidia with rough and echinulate walls ( Figure 3I , by SEM; Figure S9B ). B. bassiana formed a downy white colony with a powdery texture ( Figure 4A ), and the reverse was milky white ( Figure 4B ). Hyphae were septate and branched ( Figure 4C , by OM) and the subglobose conidia were 2.9–3.4 μm×1.7–2.1 μm in diameter ( Figure 4D , by OM; Figure 4F , by SEM). Conidiophores consisted of dense and spherical lateral clusters of globose to flask-shaped conidiogenous cells on top of an elongating and geniculate rachis ( Figure 4C , by OM; Figure 4E , by SEM). M. alternatus cadaver was wholly covered by B. bassiana mycelia ( Figure 4G ), and the characteristics of conidiophores ( Figure 4H , by SEM) and conidia ( Figure 4I , by SEM) on the beetle were the same as those on PDA medium and T. castaneum ( Figures S9C, D ). L. attenuatum colony was white and cottony ( Figure 5A ), and the reverse side was light yellow in the center with a white margin ( Figure 5B ). Hyphae were smooth-walled, carrying conidiophores in solitary, opposite, or verticillate, with long and sharp-tipped phialides that sometimes branched ( Figure 5C , by OM; Figure 5E ; by SEM). The conidia were cylindrical with slightly narrowed ends or elliptical sharp, and they were 3.2–4.4 μm×1.6–1.9 μm in diameter ( Figure 5D , by OM; Figure 5F , by SEM). M. alternatus cadaver was covered with a thin layer of mycelia, especially on its head and antennae ( Figure 5G ). The conidiophores with thorn-like phialides were grown from infecting mycelia on M. alternatus ( Figure 5H , by SEM) and T. castaneum ( Figure S9E ), and cylindrical to oval conidia were found attached to the cuticles of the two beetle species ( Figure 5I , by SEM; Figure S9F ). S. alboflavescens initially formed a powdery and translucent colony, which then turned yellowish brown with an irregular margin ( Figure 6A ). The reverse of the colony had a pale yellowish margin around the tawny center ( Figure 6B ). Cylindrical to slightly flask-shaped phialides were clustered or occasionally grown solitarily on the main stem or branches of the conidiophores ( Figure 6C , by OM). The conidia were globose to subglobose, 6.0–7.9 μm×5.6–6.9 μm in diameter ( Figure 6D , by OM), and arranged in chain ( Figure 6E , by SEM) with truncate base ( Figure 6F , by SEM). The mouthparts and leg base nodes of M. alternatus cadaver were covered with pale yellowish mycelia ( Figure 6G ). Clustered conidiophores with long and slim phialides were grown from M. alternatus ( Figure 6H , by SEM) and T. castaneum ( Figure S9G ) bodies. Conidia on beetle cuticles were rough-walled, having a truncate base ( Figure 6I , by SEM; Figure S9H ), similar to those on PDA medium.

In the ML phylogenetic analysis, the six-gene sequences of 65 species (including the seven entomopathogenic fungal species isolated in this study) were used to reconstruct the phylogenetic framework ( Table S1 ; Figure S10 ). A. austwickii strain HUZU6 and A. ruber strain HUZU28 were clustered with their respective reference strains and were separated from other Aspergillus species. P. citrinum strain HUZU144 clustered into the clade of P. citrinum CBS 139.45, was distinct from the other Penicillium species ( Figure S10 ). S. alboflavescens strain HUZU190 clustered well into a clade with S. alboflavescens CBS 399.34, which was distinct from the other Scopulariopsis species ( Figure S10 ). B. bassiana strain HUZU62, L. attenuatum strain HUZU100, and T. dorotheae strain HUZU218 matched to their corresponding reference strains and were less distinct from their phylogenetically related species ( Figure S10 ). The results of the multi-gene phylogenetic analysis conformed to the morphological features of the entomopathogenic fungal species.

Fusarium is an important Genus of pine pathogenic fungi with a relatively high isolation frequency in M. alternatus. Therefore, Fusarium species were included as positive controls for entomopathogenic fungi, to evaluate their potential ability to damage the host pine P. massoniana. Five fungal species, namely, F. annulatum, F. circinatum, P. citrinum, P. lilacinum, and T. dorotheae, showed dramatic increases in cellulase activity levels with incubation time. In contrast, cellulase activity levels of the insect-parasitic entomopathogens A. austwickii, B. bassiana, L. attenuatum and S. alboflavescens did not increase over time and were lower than those of the above five fungal species ( Figure 7A ). Regarding pectinase activity, all fungal species elevated their activity levels and remained stable at the end of the incubation, although these species showed variable time points to reach their peak activity ( Figure 7B ). F. annulatum, F. circinatum, and P. citrinum exhibited significantly higher pectinase activity than B. bassiana and S. alboflavescens, on days 8 and 10. With the exception of A. austwickii, which expressed the highest level of pectinase, the other three insect-parasitic entomopathogens showed almost the lowest pectinase activity among all fungi ( Figure 7B ).

Lesion lengths caused by fungal associates on P. massoniana seedlings further demonstrated lower pine pathogenicity of insect-parasitic entomopathogenic fungi than the other fungal species ( Figure 7C ; Kruskal-Wallis test; χ₂₀ ² = 59.80, P< 0.0001). One insect-parasitic entomopathogenic fungus, S. alboflavescens, slightly induced necrosis in the cambial zone of host pine, while exhibiting the lowest cellulase and pectinase activities among all fungi. A. austwickii had high pectinase activity but did not cause obvious lesions on the pine. Some fungal associates isolated from M. alternatus, such as F. polyphialidicum in Genus Fusarium, P. lilacinum, and P. citrinum, induced significantly greater lesions than the mock inoculation control ( Figure 7C ).