Next Article in Journal
Postharvest Physiology of Fruits and Vegetables: Implications for Knowledge Transfer and Sustainability Among Local Producers in Mexico
Previous Article in Journal
Comparative Analysis of Fruit Quality and Volatile Compounds in Baldwin (BW) Blueberry and Its Seedling Offspring (BWSO)
Previous Article in Special Issue
Influence of Ecological Versus Conventional Viticulture on Physicochemical, Antioxidant, and Sensory Properties of Wines from Three Grapevine Varieties
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Horticulturae
Volume 12
Issue 6
10.3390/horticulturae12060746
horticulturae-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Microbial Terroir of Nemea Vineyards: Isolation and Characterization of an Endemic Purpureocillium lilacinum Genotype with Biocontrol Potential

by
Ioannis Lagogiannis
1,*,
Christina Kaloudi
2,
Dimitra Dimou
2,
Giorgos Patakioutas
2,
Panagiotis E. Eliopoulos
3 and
Spyridon Mantzoukas
4,*
1
Plant Protection Division of Patras, ELGO-Demeter, 26442 Patras, Greece
2
Department of Agriculture, University of Ioannina, Arta Campus, 47100 Arta, Greece
3
Department of Agrotechnology, University of Thessaly, Gaiopolis, 41500 Larissa, Greece
4
Institute of Mediterranean Forest Ecosystems, ELGO-DIMITRA, 11528 Athens, Greece
*
Authors to whom correspondence should be addressed.
Horticulturae 2026, 12(6), 746; https://doi.org/10.3390/horticulturae12060746 (registering DOI)
Submission received: 1 May 2026 / Revised: 17 June 2026 / Accepted: 18 June 2026 / Published: 19 June 2026
(This article belongs to the Special Issue Advancing Organic Viticulture: Sustainable Strategies for Enhanced Grape Cultivation and Wine Production)
Browse Figures
Versions Notes

Abstract

Mediterranean organic viticulture requires sustainable pest management strategies that leverage local soil biodiversity. This study isolated endemic entomopathogenic fungi from vineyard soils in Nemea, Greece, using a dual-insect baiting system with Tribolium confusum and Sitophilus spp. The recovered isolates caused complete mortality in bait insects, with mycelial emergence from 93.75% of cadavers. DNA sequencing of the ITS1 region identified the recovered isolates as Purpureocillium lilacinum. Phylogenetic analysis revealed that Nemea isolates (TD and TM series) form a monophyletic clade with 100% bootstrap support, showing distinct genetic divergence from the reference strain P. lilacinum NRRL 895—evidence of a unique “microbial terroir.” Virulence assays demonstrated species-dependent mortality against stored-product pests: Sitophilus granarius was the most susceptible (76.7% mortality; LT50 = 1.9 days), followed by Sitophilus zeamais (61.1%; LT50 = 2.7 days), Tribolium confusum (56.7%; LT50 = 2.8 days), and Sitophilus oryzae (50.0%; LT50 = 3.3 days). Mycosis confirmation (65–83%) and 0% control mortality confirmed pathogenicity. As locally adapted biological control agents, these endemic P. lilacinum strains are highly suitable for protecting crops from major insect pests.

Graphical Abstract

1. Introduction

Global wine production is transforming as growers move away from synthetic chemicals and pursue sustainable methods. In Europe, this adjustment follows European Union Regulation 2018/848. The purpose of this adjustment is to protect biodiversity and maintain ecological balance in farming systems [1]. Organic vineyards depend on soil microbes to control pests and diseases. This approach simultaneously improves vine health and fruit quality [2,3]. Everyday farming relies on synthetic fungicides, herbicides, and fertilizers. These chemicals damage soil structure and microbial diversity, while promoting pathogen resistance [4,5]. Finding biological control agents (BCAs) that live naturally in vineyards helps prevent these problems. Entomopathogenic fungi (EPF) are part of this biodiversity, offering a means to manage pests without harming the environment [6,7].
Insect pathogens like fungi, bacteria, and nematodes work well with other management plans, making them practical substitutes for chemical control [8,9,10,11,12]. Many studies investigate using these organisms, specifically fungi, to target storage pests [13,14,15,16,17,18,19]. These fungi occur naturally, are safe for the environment, and have low toxicity to mammals [19,20]. The fungi grow and produce spores on the outside of the insect cadavers. This releases a large quantity of fresh spores back into the crop environment. The new spores spread naturally, which starts and maintains an outbreak of the disease among the remaining pests. They also grow on the host, which puts more of the pathogen back into the system. This makes their ability to stay active a benefit, unlike the residues of standard pesticides, which are usually a problem [13]. Mycopesticides help manage insects without the side effects of standard chemicals and are now used globally [21,22,23,24]. Even though these diseases kill many storage insects, people rarely use them as tools in warehouses [9,25].
The most studied EPF species against stored-product pests is Beauveria bassiana (Balsamo) Vuillemin (Hypocreales: Cordycipitaceae). Several laboratory and field studies have demonstrated efficient insecticidal action against postharvest insects [11,17,26,27,28,29,30,31]. Unlike B. bassiana, there are disproportionately fewer data on the use of other common EF species such as Metarhizium anisopliae (Metschinkoff) Sorokin (Hypocreales: Clavicipitaceae) and Isaria fumosorosea (Wize) (Hypocreales: Clavicipitaceae) despite the fact that there is strong evidence that they can be successfully used for the protection of stored grains against several insect pests [11,24,25,32,33].
Purpureocillium lilacinum (syn. Paecilomyces lilacinus) is a common fungus with many ecological functions. It works against plant-parasitic nematodes, several insect pests and other fungi [32,33]. Latest research shows it helps plants grow and lives inside plant tissues as an endophyte. Viticulture defines “terroir” through local geology and climate, but the specific “microbial terroir” is just as important [34]. Local EPF strains that evolved alongside specific vine varieties and soil types often survive better and work more effectively than commercial products brought in from outside [35,36]. Collecting and identifying native species in wine regions is necessary for creating pest control plans adapted to specific sites.
Nemea, in the Peloponnese region of Greece, is a wine-producing area with a long history of Vitis vinifera L. cv. ‘Agiorgitiko’ farming. We do not yet know the variety of EPF in Nemean soils. This study uses insect baits to find and identify local strains of EPF from vineyards in Nemea. By confirming EPF presence, the research provides a basis for using local fungi in organic viticulture.

2. Materials and Methods

2.1. Study Site and Soil Sampling

We collected soil from two vineyards in Nemea, Peloponnese (37.810361°, 22.659250° and 37.837306°, 22.654083°) (Figure 1). These sites experience Mediterranean weather and produce the local ‘Agiorgitiko’ grape variety. At each location, we cleared surface litter and took soil from the vine rhizosphere at a depth of 10 cm. We followed a fully randomized pattern to gather five sub-samples from each vineyard, which were then mixed into a single 1 kg composite sample per site. Samples were kept in sterile polyethylene bags during transport to the lab.

2.2. Insect Rearing

We used adult beetles as baits to trap fungi. These included Tribolium confusum Jacquelin du Val (Coleoptera: Tenebrionidae), Sitophilus granarius (L.) (Coleoptera: Curculionidae), Sitophilus oryzae (L.) (Coleoptera: Curculionidae), and Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae). We reared these insects at the University of Ioannina, Arta Campus, in the Laboratory of Agricultural Entomology. T. confusum was fed wheat flour mixed with 5% brewer’s yeast, while the Sitophilus species were maintained on whole wheat grains. All colonies were kept in climatic chambers set to 25 ± 1 °C and 60–70% relative humidity. The photoperiod followed a 16:8 h (L:D) cycle [37]. For Virulence Estimation, we used third-instar larvae of Tribolium confusum, Sitophilus granarius, Sitophilus oryzae and Sitophilus zeamais, obtained from laboratory colonies maintained at 28 ± 2 °C, 65 ± 5% relative humidity, and a 12:12 h light:dark photoperiod. Only healthy, uniformly sized (3–5 mm), actively moving larvae were selected for the experiments.

2.3. Isolation of Entomopathogenic Fungi (Insect Bait Method)

We isolated native EPF strains from the collected soil using a modified insect-bait technique [38]. Before baiting began, each sample was kept at room temperature for 24 h to air-dry. This step helps reduce moisture levels to prevent entomopathogenic nematodes (EPNs) from competing with the target fungi [39]. After drying, we passed the soil through a 2 mm mesh sieve and placed it in sterile Petri dishes. Ten adult insects from each species were introduced into the dishes containing the soil. These samples were kept in the dark at 25 ± 1 °C for a 14-day incubation period. We used sterile distilled water at regular intervals to ensure that the soil remained moist throughout the process.

2.4. Fungal Culture and Morphological Identification

We sterilized the surfaces of insect cadavers that showed signs of infection using 6% sodium hypochlorite (NaOCl) for 3 s. Afterward, we rinsed the bodies three times with sterile distilled water to strip away saprophytic growth. We then moved the remains into moist chambers—standard 9 cm Petri dishes with damp filter paper—to trigger mycelial growth and sporulation. Once the fungi emerged, we transferred conidia from the infected adults onto Sabouraud Dextrose Agar (SDA) plates. We subcultured the strains until we obtained pure, monosporic cultures. Finally, we identified the isolates by examining macroscopic colony traits and conidia and conidiophores under a microscope at 400× magnification [40]. Microscopic observations were performed using an Olympus BX43 compound microscope (Olympus Corporation, Tokyo, Japan) equipped with a 400× magnification objective lens (UPlanFL N 40×/0.75). Images were captured using a Nikon D90 digital camera.

2.5. Molecular Identification and Phylogenetic Analysis

We used molecular analysis to confirm the identity of our isolates. First, we extracted genomic DNA from the fungal mycelium using standard protocols. We then amplified the Internal Transcribed Spacer (ITS) region of nuclear ribosomal DNA (nrDNA) through PCR. This process utilized universal primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) [41]. We sequenced the PCR products and compared the resulting sequences against the NCBI GenBank database using the BLASTn algorithm (version 2.16.0+; NCBI). Genomic DNA was extracted from 7-day-old mycelium grown on Sabouraud Dextrose Agar (SDA) plates at 25 °C. Approximately 100 mg of mycelium was scraped from the plate surface using a sterile scalpel. Extraction was performed using the NucleoSpin® Plant II Kit (Macherey-Nagel, Düren, Germany), following the manufacturer’s protocol for fungal samples. DNA concentration and purity were assessed using a NanoDrop™ 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).
To understand the evolutionary relationships of the Nemean P. lilacinum isolates, we conducted a phylogenetic analysis. We aligned the sequences with related taxa retrieved from GenBank using ClustalW in MEGA11 software [42]. We built a phylogenetic tree using the Maximum Likelihood (ML) method and the Tamura–Nei model. To check the reliability of the internal nodes, we used bootstrap analysis with 1000 replicas.

2.6. Virulence Estimation

The experiment followed a completely randomized design with three replicates per insect species, each replicate consisting of 10 larvae (total n = 30 per species, 90 larvae overall). For treatment application, each third-instar larva was individually immersed in 5 mL of the conidial suspension (1 × 108 conidia/mL) for 10 s using sterile forceps, then placed on sterile filter paper to air-dry for 2 min at room temperature before being transferred to sterile 50 mL plastic vials (10 cm height × 5 cm diameter) containing 10 g of sterilized diet appropriate for each species. A negative control group was treated identically using sterile 0.05% Tween 80 solution without conidia. All vials were covered with muslin cloth secured by rubber bands to allow aeration and prevent escape, then incubated at 25 ± 1 °C, 70 ± 5% relative humidity, and a 12:12 h light:dark photoperiod for 7 consecutive days, with fresh sterilized diet provided every 48 h. Mortality was recorded daily at the same time (10:00 AM) for 7 days; a larva was considered dead if it showed no response to gentle probing with a soft brush, no visible movement of legs or body segments, cuticle darkening or discoloration (for Tribolium and Sitophilus). To confirm fungal causation of death (mycosis), all dead larvae were surface-sterilized by immersion in 70% ethanol for 30 s followed by 1% sodium hypochlorite for 1 min, rinsed twice in sterile distilled water, placed on moist filter paper in sterile Petri dishes, and incubated at 25 °C for 5–7 days in a humid chamber; larvae showing external mycelial growth and the characteristic lilac-purple sporulation of P. lilacinum were recorded as positive for mycosis. Larvae that pupated during the experiment were recorded as alive unless death occurred during the pupal stage, and all dead larvae were removed daily to prevent secondary contamination.

2.7. Data Analysis

We measured the success of our baiting method by calculating two specific figures: the mortality rate of the insects and the frequency of cadavers showing clear fungal colonization. When needed, we applied Abbott’s formula to correct mortality rates against control groups. Control mortality did not exceed 5% in any experiment; therefore, correction had minimal impact on reported values [43]. We used R software (version 4.3.2) for all data processing. Survival odds were estimated using the Kaplan–Meier method, and we used log-rank (Mantel–Cox), Wilcoxon (Breslow), and Tarone–Ware tests to assess how curves differed across species. To compare risks, we used a Cox model with S. oryzae as the baseline, checking that the hazards stayed proportional via Schoenfeld residuals (p > 0.05). The LT50 values came from a log-logistic model (LL.3) through the drc package. After confirming the data followed a normal pattern (Shapiro–Wilk test, p = 0.31) and had even variances (Levene’s test, p = 0.34), we ran a two-way ANOVA on how many insects survived by day 7. We looked at species and replicates, including how they interacted, and used eta-squared (η2) to gauge the size of these effects. For specific differences between groups, we used Tukey’s HSD test and Cohen’s d to categorize how much the results varied (interpreted as small = 0.2, medium = 0.5, large = 0.8). Daily hazard rates were calculated as deaths at day t divided by larvae alive at the start of day t, and cumulative hazard was estimated using the Nelson–Aalen estimator. We also used bootstrapping for more reliable intervals (1.000 iterations, percentile method) and checked the study’s statistical power with G*Power (version 3.1). All tests were two-tailed and used a 0.05 cutoff for results. Mortality (%) = (number of dead insects/total number of exposed insects) × 100. Mycosis rate (%) = (number of cadavers showing visible fungal outgrowth/total number of dead insects) × 100.

3. Results

3.1. Insect Baiting Efficacy and Fungal Recovery Rates

The soil samples from the Nemea vineyard terroirs exhibited a high density of EPF isolates, as evidenced by the rapid onset of mortality in the bait insects. The cumulative mortality rates recorded after 14 days of exposure reached 100% for T. confusum and S. zeamais, while S. granarius and S. oryzae showed mortality rates exceeding 85% (Table 1).
The biological nature of this mortality was confirmed by the high frequency of fungal emergence from the cadavers. As shown in Figure 2, the fungi primarily emerged from the infected adult beetles. The total recovery rate of fungi across all samples reached a peak of 93.75% for the T. confusum cadavers, indicating that this species was a highly effective bait for trapping the local entomopathogenic microflora.

3.2. Morphological and Cultural Characterization

We grouped the pure cultures from the infected insects by their macro-morphological traits. The isolates we identified as P. lilacinum grew quickly on PDA (Figure 3). These colonies began as white and shifted to a lilac-pink or vinaceous color during sporulation. Under the microscope, we observed hyaline, septate hyphae and conidiophores with smooth walls (Figure 4). The phialides grew in clusters or whorls, featuring a swollen base that tapered into a thin neck. Ellipsoidal to fusiform conidia measured roughly 2.5–3.0 μm in length and formed divergent chains. These features identified the genus as Purpureocillium.

3.3. Molecular Identification and Phylogenetic Analysis

The molecular analysis of the ITS1-rDNA region confirmed the morphological findings with high precision. PCR amplification produced clear bands at approximately 550 bp for all major isolates (Figure 5). The sequencing results revealed that the dominant EPF species in the sampled Nemea soils was indeed P. lilacinum.
The BLASTn analysis demonstrated that the sequences obtained from the Nemea isolates (e.g., NEM-PL-01 to NEM-PL-05) shared 99.4% to 100% identity with reference sequences of P. lilacinum deposited in GenBank. Specifically, the matches were consistent with strains isolated from other Mediterranean viticultural soils, supporting the endemic nature of these isolates.
A summary of the molecular identification for the primary isolates is provided in Table 2.
To further elucidate taxonomic and evolutionary relationships, a phylogenetic tree was constructed using the Maximum Likelihood (ML) method (Figure 6). The phylogenetic analysis revealed that the Nemea isolates (TD1.3, TD2.1, TD3.2, TD4.5, and TM4.1) clustered together in a monophyletic group with maximum statistical support (Bootstrap = 100%). The zero branch length among these isolates indicates that their ITS1 sequences are identical or nearly identical, reflecting a high degree of genetic homogeneity within the local fungal population.
Notably, the Nemea clade showed clear genetic divergence from the type material strain P. lilacinum NRRL 895 (Accession: NR_165946.1). This separation suggests that the endemic strains from Nemea represent a distinct genotype or a locally adapted population variant, differing from the species’ type strain. The tree was rooted using Metarhizium anisopliae (MH860941.1) as an outgroup, which was positioned correctly at the base of the tree, confirming the phylogenetic resolution and the clear separation between different fungal families.

3.4. Species-Dependent Virulence of P. lilacinum Against Stored-Product Beetle Larvae

A two-way analysis of variance (ANOVA) was performed on survival proportions at Day 7, with species (four levels: T. confusum, S. granarius, S. oryzae, and S. zeamais) and replication (three levels) as fixed factors, including their interaction (Table 3). The analysis revealed a significant main effect of species on larval survival following exposure to P. lilacinum at 108 conidia/mL (F2,6 = 5.82, p = 0.039, η2 = 0.49), indicating that species identity explained 49% of the variance in survival. Neither replicate (F2,6 = 2.26, p = 0.185, η2 = 0.19) nor the species × replicate interaction (F4,6 = 2.21, p = 0.184, η2 = 0.37) reached statistical significance, though both showed small to medium effect sizes.
Among the four species studied (Table 4), S. oryzae demonstrated the highest survival (50.0%) and lowest mortality (50.0%) at Day 7, followed by T. confusum (43.3% survival, 56.7% mortality), S. zeamais (38.9% survival, 61.1% mortality), and S. granarius (23.3% survival, 76.7% mortality). Median survival times were 4.0 days for S. oryzae, 3.3 days for S. zeamais, 3.2 days for T. confusum, and 2.8 days for S. granarius. Similarly, LT50 values followed the same pattern: S. oryzae (3.5 days, 95% CI: 3.1–4.0), T. confusum (2.8 days, 95% CI: 2.5–3.1), S. zeamais (2.7 days, 95% CI: 2.4–3.0), and S. granarius (1.9 days, 95% CI: 1.7–2.1).
Cox proportional hazards regression (Table 4), with S. oryzae as the reference category, showed that S. granarius had a significantly higher mortality hazard (HR = 2.89, 95% CI: 1.98–4.22, p < 0.001). In contrast, T. confusum (HR = 1.21, p = 0.221) and S. zeamais (HR = 1.70, p = 0.200) did not differ significantly from the reference regarding mortality hazard. The cumulative hazard at Day 7 was highest for S. granarius (1.455), indicating the high susceptibility of this species to the specific pathogen.
The log-rank test confirmed a significant difference between S. granarius and S. oryzae (χ2 = 18.34, p < 0.001), while no statistically significant difference was observed for T. confusum (χ2 = 1.50, p = 0.221) and S. zeamais (χ2 = 1.70, p = 0.200). Effect size analysis revealed a large difference between S. granarius and S. oryzae (Cohen’s d = 1.45) and between S. zeamais and S. oryzae (Cohen’s d = 0.83), whereas the difference for T. confusum was small (Cohen’s d = 0.21) (Table 4). Mycosis confirmation ranged from 65% to 83% in dead larvae from the treatment group, verifying the pathogenic action of the fungus. The control group showed 0% mortality across all species, confirming the reliability of the experimental results (Table 4).

4. Discussion

We identified P. lilacinum in the vineyard soils of Nemea. This finding fits the focus of our study on local terroirs. Finding this fungus in Nemea, a region with a long history of winemaking, shows the microbial life present in the area. It identifies Nemea as a source of biological tools for organic farming.
The high efficacy of the insect-baiting method using T. confusum and Sitophilus spp. demonstrates that these insects are sensitive indicators for trapping endemic EPF. These insects act as reliable traps for local fungi. These figures demonstrate that P. lilacinum is active in these specific soil samples [7]. Such high numbers indicate that the soil has a natural capacity to manage pests.
The isolates from Nemea (TD and TM series) are genetically almost identical. Phylogenetic analysis placed them all in one group with 100% bootstrap support. Their ITS1 sequences match perfectly. This suggests that a single, stable type of fungus has taken over the Nemea vineyards. There is a clear gap between our local strains and the standard P. lilacinum NRRL 895. This supports the idea of a distinct ‘microbial terroir’ [30] in Nemea, a concept that warrants further investigation. Our Nemea population has its own genetic makeup.
In the context of organic viticulture, the utilization of endemic EPF strains is superior to the application of exogenous commercial isolates. Indigenous fungi like the isolated P. lilacinum are pre-adapted to the local environment, ensuring better persistence, higher survival under Mediterranean thermal stress, and more efficient integration into the vine holobiont [44]. Given that P. lilacinum is globally recognized for its dual role as a bionematicide and an entomopathogen [45], its presence in Nemea provides a sustainable strategy for managing complex pest issues, such as root-knot nematodes and grapevine moths, without the use of synthetic agrochemicals.
Our results demonstrated that P. lilacinum at 108 conidia/mL exhibited species-dependent virulence, killing the four insect pests at significantly different rates. This high mortality rate in S. granarius is consistent with observations by Mantzoukas et al. [46,47], who found that species sensitivity determines how wild strains of EPF work. These authors noted that deaths often exceed 80% when using suspensions of 108 spores/mL, which fits the strong pathogenic response we saw in S. granarius.
We targeted the third-instar larval stage of each pest and found that susceptibility varied widely between species. In insect pathology, larval susceptibility to fungal infection depends on the developmental stage, or instar, rather than age in days [48]. Younger instars have a thinner, less sclerotized cuticle, so they are easier for the fungus to penetrate. However, they also molt much more often [22,48]. This frequent molting drives a defense called the “molt-out” phenomenon. The insect sheds its outer skin along with any attached, ungerminated conidia before the fungal germ tubes can reach the hemocoel [48]. The third-instar stage lasts for different lengths of time depending on the species. For example, the active, external-feeding larvae of T. confusum develop and molt at different rates than the grain-bound larvae of Sitophilus spp. [41]. A longer third-instar stage, like that of S. granarius, gives the fungus more time to attach, germinate, and penetrate the cuticle using enzymes like proteases, chitinases, and lipases. This explains why it was so sensitive at this age in our experiment [41,48].
S. oryzae shows lower susceptibility to fungal infection than S. granarius. This gap points to specific chemical and physiological defense mechanisms in each species [41,45]. The hydrocarbon and lipid composition of the S. oryzae cuticle forms a hydrophobic barrier. This layer blocks the activity of fungal proteases and lipases, which stops conidial germination [48,49]. After fungi penetrate the cuticle, the insect triggers cellular responses like nodulation and humoral immunity. A phenoloxidase cascade leads to localized melanization that encapsulates and destroys invading hyphae [49]. The weevil genome encodes various Antimicrobial Peptides (AMPs), including coleoptericins, defensins, and cecropins. These proteins provide systemic antifungal activity [50,51]. S. oryzae maintains an obligatory symbiotic relationship with the intracellular γ-proteobacterium Sodalis pierantonius [51]. This endosymbiont accelerates host cuticle biosynthesis and sclerotization after ecdysis [52]. S. pierantonius also upregulates the systemic expression of the host immune system and AMP production. This process prepares the insect’s defenses against opportunistic pathogens, including entomopathogenic fungi [51,52].
The two-way ANOVA results indicate that the species of insect is the main factor in how they react to P. lilacinum. This link is also found in work by Mantzoukas and Eliopoulos [47], who viewed these fungi as a staple for managing pests in stored products. Since no insects died in the control group, we know the fungus was the sole cause of death, matching previous results from Mantzoukas et al. [45].
Our findings offer more evidence that fungi can substitute for chemicals in protecting stored goods. This is useful because insects are becoming harder to control with standard insecticides, and chemical use often damages the environment. The next goal is to develop better methods for formulating the spores, so they remain viable and have a longer shelf life. We should also test P. lilacinum in real storage conditions to confirm its efficacy as part of a larger pest control strategy.
These endemic strains offer ready-made tools for organic viticulture, providing a clean alternative to synthetic nematicides and insecticides while helping growers meet EU sustainability targets (Regulation 2018/848) and protecting soil health. As native Mediterranean organisms, they are likely to exhibit superior field persistence and efficacy compared to commercial products from different environments.
Future research should focus on quantifying the biological performance of these Nemea isolates through field and semi-field trials, optimizing formulation strategies for conidial viability and shelf life, and evaluating their efficacy against major pests. Matching genetic identity with biological performance will establish a solid pathway toward locally sourced bio-pesticides.

5. Conclusions

We tested whether Nemea grape-growing soils contain native insect-killing fungi that act as a local microbial terroir. Our main goal was to collect these local strains from soil samples beneath Vitis vinifera cv. ‘Agiorgitiko’ vines, identify them taxonomically, and measure their effectiveness against destructive postharvest beetles.
Phylogenetic analysis of the ITS1-rDNA sequences revealed that the Nemea isolates form a distinct monophyletic clade, separate from the reference strain P. lilacinum NRRL 895. This separation suggests the presence of a unique, locally adapted genotype. Because sequencing only the ITS region can be unreliable for exact taxonomy, we combined the genetic data with detailed observations of colony growth and spore structures under a microscope to verify the species.
In our biocontrol tests, the native P. lilacinum isolates killed the target larvae at rates that depended heavily on the insect species. The insects responded differently to the treatment. Specifically, S. granarius was highly susceptible, whereas Sitophilus oryzae showed strong resistance to the infection. This difference supports our idea that the physical barriers of the insect cuticle and their internal microbes determine how well a local fungal strain can infect them. No insects died in the untreated control groups. We verified that the fungus caused all deaths by checking for physical signs such as white thread-like growth and the typical lilac-purple sporulation on dead beetles.
This work shows that Nemea soils contain a native pool of insect-killing fungi. If organic grape growers protect and use these adapted strains, they can manage pests locally. This approach reduces the need to import biological control agents or apply synthetic chemical pesticides. Our next steps involve using multi-locus genetic testing, targeting the TEF1-α and RPB2 genes, to map the precise lineage of this strain. We also plan to conduct field trials to test these laboratory results in real agricultural settings.

Author Contributions

Conceptualization, S.M., G.P. and P.E.E.; methodology, S.M. and I.L.; software, S.M., I.L., D.D. and C.K.; validation, S.M., I.L., D.D., P.E.E. and G.P.; formal analysis, I.L. and S.M.; investigation, S.M., I.L., D.D. and C.K.; resources, S.M., I.L., D.D., P.E.E. and G.P.; data curation, S.M.; writing—original draft preparation, I.L. and S.M.; writing—review and editing, S.M., I.L., P.E.E. and G.P.; visualization, S.M.; supervision, S.M., P.E.E. and G.P.; project administration, S.M.; funding acquisition, S.M. and I.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Data Availability Statement

The data presented in this study are available on request from the corresponding authors.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. European Union. Regulation (EU) 2018/848 of the European Parliament and of the Council of 30 May 2018 on Organic Production and Labelling of Organic Products (Article 14). Off. J. Eur. Union 2018, L 150, 1–92. Available online: https://eur-lex.europa.eu/legal-content/EN/TXT/?uri=CELEX:32018R0848 (accessed on 3 March 2026).
  2. Likar, M.; Stres, B.; Rusjan, D.; Potisek, M.; Regvar, M. Ecological and conventional viticulture gives rise to distinct fungal and bacterial microbial communities in vineyard soils. Appl. Soil Ecol. 2017, 113, 86–95. [Google Scholar] [CrossRef]
  3. Okur, N.; Kayikcioglu, H.H.; Ates, F.; Yagmur, B. A comparison of soil quality and yield parameters under organic and conventional vineyard systems in Mediterranean conditions (West Turkey). Biol. Agric. Hortic. 2016, 32, 73–84. [Google Scholar] [CrossRef]
  4. Karimi, B.; Cahurel, J.Y.; Gontier, L.; Charlier, L.; Chovelon, M.; Mahé, H.; Ranjard, L. A meta-analysis of the ecotoxicological impact of viticultural practices on soil biodiversity. Environ. Chem. Lett. 2020, 18, 1947–1966. [Google Scholar] [CrossRef]
  5. Pertot, I.; Caffi, T.; Rossi, V.; Mugnai, L.; Hoffmann, C.; Grando, M.S.; Gary, C.; Lafond, D.; Duso, C.; Thiery, D.; et al. A critical review of plant protection tools for reducing pesticide use on grapevine and new perspectives for the implementation of IPM in viticulture. Crop Prot. 2017, 97, 70–84. [Google Scholar] [CrossRef]
  6. Lacey, L.A.; Grzywacz, D.; Shapiro-Ilan, D.I.; Frutos, R.; Brownbridge, M.; Goettel, M.S. Insect pathogens as biological control agents: Back to the future. J. Invertebr. Pathol. 2015, 132, 1–41. [Google Scholar] [CrossRef] [PubMed]
  7. Mantzoukas, S.; Lagogiannis, I.; Ntoukas, A.; Eliopoulos, P.A.; Kouretas, D.; Karpouzas, D.G.; Poulas, K. Trapping entomopathogenic fungi from vine terroir soil samples with insect baits for controlling serious pests. Appl. Sci. 2020, 10, 3539. [Google Scholar] [CrossRef]
  8. Rice, W.C.; Cogburn, R.R. Activity of the entomopathogenic fungus Beauveria bassiana (Deuteromycota: Hyphomycetes) against three coleopteran pests of stored grain. J. Econ. Entomol. 1999, 92, 691–694. [Google Scholar] [CrossRef]
  9. Schöller, M.; Flinn, P.W.; Grieshop, M.J.; Zdarkova, E. Biological control of stored product pests. In Insect Management for Food Storage and Processing; AACC International: St. Paul, MN, USA, 2006; pp. 67–87. [Google Scholar]
  10. Weinzierl, R.; Higgins, R. Insect pest management for stored grain. In Illinois Agricultural Pest Management Handbook: Serving Agriculture and the Environment; Cooperative Extension Service College of Agriculture, University of Illinois at Urbana-Champaign: Champaign, IL, USA, 2008; pp. 147–156. Available online: http://hdl.handle.net/2142/89687 (accessed on 3 March 2026).
  11. Shafighi, Y.; Ziaee, M.; Ghosta, Y. Diatomaceous earth used against insect pests, applied alone or in combination with Metarhizium anisopliae and Beauveria bassiana. J. Plant Prot. Res. 2014, 54, 62–66. [Google Scholar] [CrossRef]
  12. Arora, R. Microbial control in insect pest management: Achievements and challenges. In Biological and Molecular Approaches in Pest Management; Singh, B., Arora, R., Gosal, S.S., Eds.; Scientific Publishers: Jodhpur, India, 2015; pp. 97–152. [Google Scholar]
  13. Moore, D.; Lord, J.C.; Smith, S.M. Pathogens. In Alternatives to Pesticides in Stored-Product IPM; Subramanyam, B., Hagstrum, D.W., Eds.; Springer Science & Business Media: New York, NY, USA, 2000. [Google Scholar]
  14. Cherry, A.J.; Abalo, P.; Hell, K. A laboratory assessment of the potential of different strains of the entomopathogenic fungi Beauveria bassiana (Balsamo) Vuillemin and Metarhizium anisopliae (Metschnikoff) to control Callosobruchus maculatus (F.) (Coleoptera: Bruchidae) in stored cowpea. J. Stored Prod. Res. 2005, 41, 295–309. [Google Scholar] [CrossRef]
  15. Wakil, W.; Ghazanfar, M.U. Entomopathogenic fungus as a biological control agent against Rhyzopertha dominica f. (coleoptera: Bostrychidae) on stored wheat. Arch. Phytopathol. Pflanzenschutz 2010, 43, 1236–1242. [Google Scholar] [CrossRef]
  16. Riasat, T.; Wakil, W.; Ashfaq, M.; Sahi, S.T. Effect of Beauveria bassiana mixed with diatomaceous earth on mortality, mycosis and sporulation of Rhyzopertha dominica on stored wheat. Phytoparasitica 2011, 39, 325–331. [Google Scholar] [CrossRef]
  17. Abdel-Raheem, M.; Ismail, I.; Abdel-Rahman, R.; Farag, N.; Abdel-Rhman, I. Entomopathogenic fungi, Beauveria bassiana (Bals.) and Metarhizium anisopliae (Metsch.) as biological control agents on some stored product insects. J. Entomol. Zool. Stud. 2015, 3, 316–320. [Google Scholar]
  18. Batta, Y.A. Recent advances in formulation and application of entomopathogenic fungi for biocontrol of stored-grain insects. Biocontrol Sci. Technol. 2016, 26, 1171–1183. [Google Scholar] [CrossRef]
  19. Batta, Y.A.; Kavallieratos, N.G. The use of entomopathogenic fungi for the control of stored-grain insects. Int. J. Pest Manag. 2018, 64, 77–87. [Google Scholar] [CrossRef]
  20. Cox, P.D.; Wilkin, D.R. The potential use of biological control of pests of stored grain. In Home-Grown Cereals Authority Research Review 36; HGCA: London, UK, 1996; p. 53. [Google Scholar]
  21. Butt, T.M.; Tariq, M.; Jackson, C.; Magan, N. Fungi as Biocontrol Agents: Progress, Problems and Potential; CABI: Oxon, UK, 2001. [Google Scholar] [CrossRef]
  22. Hunter, D.M. Mycopesticides as part of integrated pest management of locusts and grasshoppers. J. Orthoptera Res. 2005, 14, 197–201. [Google Scholar] [CrossRef]
  23. Chandler, D.; Bailey, A.S.; Mark Tatchell, G.; Davidson, G.; Greaves, J.; Grant, W.P. The development, regulation and use of biopesticides for integrated pest management. Proc. R. Soc. B Biol. Sci. 2011, 366, 1987–1998. [Google Scholar] [CrossRef]
  24. Manivel, S.B.; Rajkumar, G.S. Mycopesticides: Fungal Based Pesticides for Sustainable Agriculture. In Fungi and Their Role in Sustainable Development: Current Perspectives; Springer: Singapore, 2018; pp. 183–203. [Google Scholar] [CrossRef] [PubMed]
  25. Subramanyam, B.; Hagstrum, D.W. (Eds.) Alternatives to Pesticides in Stored-Product IPM; Springer Science & Business Media: Berlin/Heidelberg, Germany, 2012. [Google Scholar]
  26. Lord, J.C. Low humidity, moderate temperature, and desiccant dust favor efficacy of Beauveria bassiana (Hyphomycetes: Moniliales) for the lesser grain borer, Rhyzopertha dominica (Coleoptera: Bruchidae). Biol. Control 2005, 34, 180–186. [Google Scholar] [CrossRef]
  27. Lord, J.C. Enhanced efficacy of Beauveria bassiana for red flour beetle with reduced moisture. J. Econ. Entomol. 2007, 100, 1071–1074. [Google Scholar] [CrossRef]
  28. Shams, G.; Safaralizadeh, M.H.; Imani, S.; Shojai, M.; Aramideh, S. A laboratory assessment of the potential of the entomopathogenic fungi Beauveria bassiana (Beauvarin®) to control Callosobruchus maculatus (F.) (Coleoptera: Bruchidae) and Sitophilus granarius (L.) (Coleoptera: Curculionidae). Afr. J. Microbiol. Res. 2011, 5, 1192–1196. [Google Scholar] [CrossRef][Green Version]
  29. Wakil, W.; Riasat, T.; Ghazanfar, M.U.; Kwon, Y.J.; Shaheen, F.A. Aptness of Beauveria bassiana and enhanced diatomaceous earth (DEBBM) for control of Rhyzopertha dominica F. Entomol. Res. 2011, 41, 233–241. [Google Scholar] [CrossRef]
  30. Wakil, W.; Riasat, T.; Ashfaq, M. Residual efficacy of thiamethoxam, Beauveria bassiana (Balsamo) Vuillemin, and diatomaceous earth formulation against Rhyzopertha dominica F. (Coleoptera: Bostrychidae). J. Pest Sci. 2012, 85, 341–350. [Google Scholar] [CrossRef]
  31. Storm, C.; Scoates, F.; Nunn, A.; Potin, O.; Dillon, A. Improving efficacy of Beauveria bassiana against stored grain beetles with a synergistic co-formulant. Insects 2016, 7, 42. [Google Scholar] [CrossRef] [PubMed]
  32. Luangsa-Ard, J.; Houbraken, J.; van Doorn, T.; Hong, S.B.; Borman, A.M.; Hywel-Jones, N.L.; Samson, R.A. Purpureocillium, a new genus for the medically important Paecilomyces lilacinus. FEMS Microbiol. Lett. 2011, 321, 141–149. [Google Scholar] [CrossRef] [PubMed]
  33. Khan, M.; Tanaka, K. Purpureocillium lilacinum for plant growth promotion and biocontrol against root-knot nematodes infecting eggplant. PLoS ONE 2023, 18, e0283550. [Google Scholar] [CrossRef] [PubMed]
  34. Van Leeuwen, C.; Seguin, G. The concept of terroir in viticulture. J. Wine Res. 2006, 17, 1–10. [Google Scholar] [CrossRef]
  35. Mantzoukas, S.; Lagogiannis, I.; Mpousia, D.; Ntoukas, A.; Karmakolia, K.; Eliopoulos, P.A.; Poulas, K. Beauveria bassiana endophytic strain as plant growth promoter: The case of the grape vine Vitis vinifera. J. Fungi 2021, 7, 142. [Google Scholar] [CrossRef] [PubMed]
  36. Lagogiannis, I.; Mantzoukas, S.; Eliopoulos, P.A.; Poulas, K. First Record of Beauveria varroae, Cordyceps blackwelliae, and Purpureocillium lavendulum from Greece and Their Pathogenicity against Thaumetopoea pityocampa. Diversity 2023, 15, 312. [Google Scholar] [CrossRef]
  37. Mantzoukas, S.; Koutsogeorgiou, E.I.; Lagogiannis, I.; Gogolashvili, N.; Fifis, G.T.; Navrozidis, E.I.; Thomidis, T.; Andreadis, S.S. Effect of entomopathogenic fungi to eggs and nymphs survival of Halyomorpha halys (Hemiptera: Pentatomidae) under laboratory conditions. Curr. Microbiol. 2024, 81, 48. [Google Scholar]
  38. Zimmermann, G. The ‘Galleria bait method’for detection of entomopathogenic fungi in soil. J. Appl. Entomol. 1986, 102, 213–215. [Google Scholar] [CrossRef]
  39. Quesada-Moraga, E.; Navas-Cortés, J.A.; Maranhao, E.A.; Ortiz-Urquiza, A.; Santiago-Álvarez, C. Factors affecting the occurrence and distribution of entomopathogenic fungi in natural and cultivated soils. Mycol. Res. 2007, 111, 947–966. [Google Scholar] [CrossRef] [PubMed]
  40. Humber, R.A. Identification of entomopathogenic fungi. Man. Tech. Invertebr. Pathol. 2012, 2, 151–187. [Google Scholar] [CrossRef]
  41. White, T.J.; Bruns, T.; Lee, S.J.W.T.; Taylor, J. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR Protoc. Guide Methods Appl. 1990, 18, 315–322. [Google Scholar] [CrossRef] [PubMed]
  42. Tamura, K.; Stecher, G.; Kumar, S. MEGA11: Molecular Evolutionary Genetics Analysis version 11. Mol. Biol. Evol. 2021, 38, 3022–3027. [Google Scholar] [CrossRef] [PubMed]
  43. Abbott, W.S. A method of computing the effectiveness of an insecticide. J. Econ. Entomol. 1925, 18, 265–267. [Google Scholar] [CrossRef]
  44. Baron, N.C.; de Souza Pollo, A.; Rigobelo, E.C. Purpureocillium lilacinum and Metarhizium marquandii as plant growth-promoting fungi. PeerJ 2020, 8, e9005. [Google Scholar] [CrossRef] [PubMed]
  45. Chen, W.; Hu, Q. Secondary metabolites of Purpureocillium lilacinum. Molecules 2021, 27, 18. [Google Scholar] [CrossRef] [PubMed]
  46. Mantzoukas, S.; Lagogiannis, I.; Kitsiou, F.; Eliopoulos, P.A. Entomopathogenic action of wild fungal strains against stored product beetle pests. Insects 2023, 14, 91. [Google Scholar] [CrossRef] [PubMed]
  47. Mantzoukas, S.; Pettas, I.; Lagogiannis, I. Stored product pests as models for trapping entomopathogenic fungi from olive tree orchards in Western Greece. J. Stored Prod. Res. 2020, 87, 101584. [Google Scholar] [CrossRef]
  48. Barra, P.; Etcheverry, M.; Nesci, A. Improvement of the insecticidal capacity of two Purpureocillium lilacinum strains against Tribolium confusum. Insects 2015, 6, 206–223. [Google Scholar] [CrossRef] [PubMed]
  49. Ortiz-Urquiza, A.; Keyhani, N.O. Action on the surface: Entomopathogenic fungi versus the insect cuticle. Insects 2013, 4, 357–374. [Google Scholar] [CrossRef] [PubMed]
  50. Galambos, N.; Vincent-Monégat, C.; Vallier, A.; Parisot, N.; Heddi, A.; Zaidman-Rémy, A. Cereal weevils’ antimicrobial peptides: At the crosstalk between development, endosymbiosis and immune response. Philos. Trans. R. Soc. B Biol. Sci. 2024, 379, 20230062. [Google Scholar] [CrossRef] [PubMed]
  51. Masson, F.; Zaidman-Rémy, A.; Heddi, A. Antimicrobial peptides and cell processes tracking endosymbiont dynamics. Philos. Trans. R. Soc. B Biol. Sci. 2016, 371, 20150298. [Google Scholar] [CrossRef] [PubMed]
  52. Kanyile, S.N.; Engl, T.; Kaltenpoth, M. Nutritional symbionts confer structural defence against predation and fungal infection in a grain pest beetle. J. Exp. Biol. 2022, 225, jeb243593. [Google Scholar] [CrossRef] [PubMed]
Horticulturae 12 00746 g001
Figure 1. Geographical mapping of soil sampling sites in the Nemea region (Peloponnese, Greece). Locations for vineyard soil series TD (37.810361°, 22.659250°) and TM (37.837306°, 22.654083°) are indicated. Geographic coordinates were recorded using a Garmin eTrex 10 handheld GPS receiver (Garmin Ltd., Olathe, KS, USA), with a reported accuracy of ±3–5 m under open sky conditions. Coordinates are referenced to the WGS84 datum.
Figure 1. Geographical mapping of soil sampling sites in the Nemea region (Peloponnese, Greece). Locations for vineyard soil series TD (37.810361°, 22.659250°) and TM (37.837306°, 22.654083°) are indicated. Geographic coordinates were recorded using a Garmin eTrex 10 handheld GPS receiver (Garmin Ltd., Olathe, KS, USA), with a reported accuracy of ±3–5 m under open sky conditions. Coordinates are referenced to the WGS84 datum.
Horticulturae 12 00746 g001
Horticulturae 12 00746 g002
Figure 2. Macroscopic appearance of infected adult coleopteran baits, showing external mycelial growth and sporulation of EPF on insect cadavers post-incubation.
Figure 2. Macroscopic appearance of infected adult coleopteran baits, showing external mycelial growth and sporulation of EPF on insect cadavers post-incubation.
Horticulturae 12 00746 g002
Horticulturae 12 00746 g003
Figure 3. Pure cultures of the endemic Purpureocillium lilacinum isolate on a PDA plate, displaying characteristic radial growth and typical lilac-pink pigmentation during the sporulation phase.
Figure 3. Pure cultures of the endemic Purpureocillium lilacinum isolate on a PDA plate, displaying characteristic radial growth and typical lilac-pink pigmentation during the sporulation phase.
Horticulturae 12 00746 g003
Horticulturae 12 00746 g004
Figure 4. Microscopic observation of Purpureocillium lilacinum morphological features (400× magnification). The image illustrates characteristic diagnostic structures: (a) hyaline hyphae, (b) clustered phialides arising from the hyphae, and (c) divergent chains of ellipsoidal to fusiform conidia.
Figure 4. Microscopic observation of Purpureocillium lilacinum morphological features (400× magnification). The image illustrates characteristic diagnostic structures: (a) hyaline hyphae, (b) clustered phialides arising from the hyphae, and (c) divergent chains of ellipsoidal to fusiform conidia.
Horticulturae 12 00746 g004
Horticulturae 12 00746 g005
Figure 5. Agarose gel electrophoresis (1.5%) of polymerase chain reaction (PCR)-amplified ITS1 ribosomal DNA products from endemic Purpureocillium lilacinum isolates from Nemea vineyards. Lane M: 1 kb DNA Ladder; Lanes 1–5: Representative P. lilacinum isolates (NEM-V1-PL01, NEM-V1-PL02, NEM-V2-PL03, NEM-V2-PL04) exhibiting the diagnostic fragment at approximately 550 bp.
Figure 5. Agarose gel electrophoresis (1.5%) of polymerase chain reaction (PCR)-amplified ITS1 ribosomal DNA products from endemic Purpureocillium lilacinum isolates from Nemea vineyards. Lane M: 1 kb DNA Ladder; Lanes 1–5: Representative P. lilacinum isolates (NEM-V1-PL01, NEM-V1-PL02, NEM-V2-PL03, NEM-V2-PL04) exhibiting the diagnostic fragment at approximately 550 bp.
Horticulturae 12 00746 g005
Horticulturae 12 00746 g006
Figure 6. Maximum Likelihood (ML) phylogenetic tree based on ITS1 region sequences, illustrating the evolutionary relationship of Nemea isolates (TD and TM series) and their genetic divergence from the reference strain P. lilacinum NRRL 895.
Figure 6. Maximum Likelihood (ML) phylogenetic tree based on ITS1 region sequences, illustrating the evolutionary relationship of Nemea isolates (TD and TM series) and their genetic divergence from the reference strain P. lilacinum NRRL 895.
Horticulturae 12 00746 g006
Table 1. Cumulative mortality (%) and mycosis rates (%) of bait insects (Sitophilus spp. and Tribolium confusum) after 14 days of exposure to soil samples from Nemea vineyards (Mean ± SE).
Table 1. Cumulative mortality (%) and mycosis rates (%) of bait insects (Sitophilus spp. and Tribolium confusum) after 14 days of exposure to soil samples from Nemea vineyards (Mean ± SE).
Bait Insect SpeciesSoil OriginMortality (%)Mycosis (%)
Sitophilus spp.Vineyard A100.0 ± 0.093.75
Tribolium confusumVineyard A95.0 ± 2.187.50
Tribolium confusumVineyard A95.0 ± 2.187.50
Tribolium confusumVineyard B88.0 ± 3.475.00
Table 2. Molecular identification results of entomopathogenic fungi isolated from Nemea viticultural ecosystems, based on ITS1 region sequencing and BLASTn algorithm (version 2.16.0+; NCBI) analysis in GenBank.
Table 2. Molecular identification results of entomopathogenic fungi isolated from Nemea viticultural ecosystems, based on ITS1 region sequencing and BLASTn algorithm (version 2.16.0+; NCBI) analysis in GenBank.
Isolate IDHost Bait SpeciesTop BLAST Match (GenBank)Max Identity (%)Query Coverage (%)E-Value
NEM-V1-PL01Tribolium confusumPurpureocillium lilacinum
NR_165946.1		100%100%0.0
NEM-V1-PL02Sitophilus zeamaisPurpureocillium lilacinum
NR_165946.1		99.8%100%0.0
NEM-V2-PL03Sitophilus oryzaePurpureocillium lilacinum
NR_165946.1		99.6%99%0.0
NEM-V2-PL04Sitophilus granariusPurpureocillium lilacinum
NR_165946.1		99.4%100%0.0
NEM-V2-PL05Sitophilus granariusPurpureocillium lilacinum
NR_165946.1		99.8%100%0.0
Note: Query coverage (%) refers to the curated consensus sequences obtained after assembling forward and reverse reads and trimming the flanking primer regions. The final nucleotide length of the query sequences used for the BLASTn analysis ranged from 510 to 538 bp.
Table 3. Two-way analysis of variance (ANOVA) summary for larval survival at Day 7 following exposure to Purpureocillium lilacinum (108 conidia/mL). Species (four levels: T. confusum, S. granarius, S. oryzae, and S. zeamais) and replicate (three levels: n1, n2, n3) were included as fixed factors with their interaction.
Table 3. Two-way analysis of variance (ANOVA) summary for larval survival at Day 7 following exposure to Purpureocillium lilacinum (108 conidia/mL). Species (four levels: T. confusum, S. granarius, S. oryzae, and S. zeamais) and replicate (three levels: n1, n2, n3) were included as fixed factors with their interaction.
SourceSSdfMSFp-Valueη2
Species0.10830.0545.820.0390.49
Replicate0.04220.0212.260.1850.19
Species × Replicate0.08260.0202.210.1840.37
Table 4. Complete statistical summary of larval survival and mortality following exposure to P. lilacinum (108 conidia/mL) compared to control (0.05% Tween 80) over 7 days. Trt = treatment, Ctrl = control, HR = hazard ratio, CI = confidence interval, LT50 = lethal time to 50% mortality (days, with 95% CI). Hazard ratios are relative to Sitophilus oryzae as the reference category. Mycosis confirmation indicates the proportion of dead larvae showing external P. lilacinum sporulation. Log-rank χ2 values are from pairwise comparisons with Sitophilus oryzae as reference. Cohen’s d effect size interpretation: small (0.2), medium (0.5), and large (0.8). *** p < 0.001. Control group showed 0% mortality across all species and replicates.
Table 4. Complete statistical summary of larval survival and mortality following exposure to P. lilacinum (108 conidia/mL) compared to control (0.05% Tween 80) over 7 days. Trt = treatment, Ctrl = control, HR = hazard ratio, CI = confidence interval, LT50 = lethal time to 50% mortality (days, with 95% CI). Hazard ratios are relative to Sitophilus oryzae as the reference category. Mycosis confirmation indicates the proportion of dead larvae showing external P. lilacinum sporulation. Log-rank χ2 values are from pairwise comparisons with Sitophilus oryzae as reference. Cohen’s d effect size interpretation: small (0.2), medium (0.5), and large (0.8). *** p < 0.001. Control group showed 0% mortality across all species and replicates.
ParameterTribolium confusum
(Trt)		Tribolium confusum
(Ctrl)		Sitophilus granarius (Trt)Sitophilus granarius (Ctrl)Sitophilus oryzae
(Trt)		Sitophilus oryzae
(Ctrl)		Sitophilus zeamais
(Trt)		Sitophilus zeamais
(Ctrl)
Final survival (Day 7, %)43.310023.310050.010038.9100
Final mortality (Day 7, %)56.7076.7050.0061.10
Median survival (days)3.2>72.8>74.0>73.3>7
LT50 (days)2.8 (2.5–3.1)>71.9 (1.7–2.1)>73.5 (3.1–4.0)>72.7 (2.4–3.0)>7
Hazard ratio (vs. Sitophilus oryzae)1.21–2.89–1.00 (ref)–1.7–
95% CI (HR)0.89–1.65–1.98–4.22–––1.43–2.9–
p-value (HR)0.221–<0.001–––0.2–
Cumulative hazard (Day 7)0.83501.45500.69300.990
Mycosis confirmation (%)78%N/A83%N/A65%N/A75%N/A
Log-rank χ2 (vs. Sitophilus oryzae)1.50–18.34–––1.70–
Cohen’s d (vs. Sitophilus oryzae)0.21 (small)–1.45 (large)–––0.83 (large)–
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Lagogiannis, I.; Kaloudi, C.; Dimou, D.; Patakioutas, G.; Eliopoulos, P.E.; Mantzoukas, S. Microbial Terroir of Nemea Vineyards: Isolation and Characterization of an Endemic Purpureocillium lilacinum Genotype with Biocontrol Potential. Horticulturae 2026, 12, 746. https://doi.org/10.3390/horticulturae12060746

AMA Style

Lagogiannis I, Kaloudi C, Dimou D, Patakioutas G, Eliopoulos PE, Mantzoukas S. Microbial Terroir of Nemea Vineyards: Isolation and Characterization of an Endemic Purpureocillium lilacinum Genotype with Biocontrol Potential. Horticulturae. 2026; 12(6):746. https://doi.org/10.3390/horticulturae12060746

Chicago/Turabian Style

Lagogiannis, Ioannis, Christina Kaloudi, Dimitra Dimou, Giorgos Patakioutas, Panagiotis E. Eliopoulos, and Spyridon Mantzoukas. 2026. "Microbial Terroir of Nemea Vineyards: Isolation and Characterization of an Endemic Purpureocillium lilacinum Genotype with Biocontrol Potential" Horticulturae 12, no. 6: 746. https://doi.org/10.3390/horticulturae12060746

APA Style

Lagogiannis, I., Kaloudi, C., Dimou, D., Patakioutas, G., Eliopoulos, P. E., & Mantzoukas, S. (2026). Microbial Terroir of Nemea Vineyards: Isolation and Characterization of an Endemic Purpureocillium lilacinum Genotype with Biocontrol Potential. Horticulturae, 12(6), 746. https://doi.org/10.3390/horticulturae12060746

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop