Native Fungus Wipes Out Mustard Aphids and Survives Brutal Heat in Lab Tests

A fungus collected from northern Thai soils has emerged as a standout candidate against one of the most damaging pests of cruciferous crops. In a laboratory study published in Frontiers in Fungal Biology, researchers screened ten indigenous isolates of the entomopathogenic fungus Metarhizium anisopliae against the mustard aphid, Lipaphis erysimi, and identified one strain, MaCMUIPL 57, that combined rapid lethality with unusually strong sporulation and heat tolerance. The work, led by Sarayut Pittarate and colleagues at Chiang Mai University with collaborators at National Chung Hsing University in Taiwan, integrates pathogenicity testing, phenotypic profiling, and molecular analysis in a single evaluation framework for selecting biocontrol agents.

The mustard aphid is a serious threat to brassicaceous crops across tropical and subtropical regions, attacking Chinese kale, mustard, and cabbage through continuous phloem feeding. According to the authors, aphid-induced yield losses can reach 50 percent and occasionally 100 percent when the insects vector viral diseases, while current management depends heavily on synthetic insecticides that drive resistance, pest resurgence, and environmental contamination. Entomopathogenic fungi offer an alternative because they infect insects directly through the cuticle, self-propagate, and fit within integrated pest management programs, but their field performance varies considerably among isolates, particularly where temperatures and humidity fall outside the narrow range in which these fungi grow best.

The ten isolates, labeled MaCMUIPL 2 through MaCMUIPL 57, were originally recovered from soil samples in natural forests and agricultural lands using the insect bait method with larvae of the mealworm Tenebrio molitor. In a preliminary contact plate assay, all ten killed model T. molitor larvae completely within three to four days. The team then tested pathogenicity against second-instar mustard aphid nymphs at a single concentration of 1 × 10⁶ conidia per milliliter using a leaf-disc dipping method, with each treatment replicated four times and mortality recorded daily for 14 days. Five isolates, MaCMUIPL 14, 37, 40, 43, and 57, achieved 100 percent aphid mortality within seven days after inoculation, while the remaining isolates caused 78 to 98 percent mortality over the same period.

Timing distinguished the top performers. MaCMUIPL 57 killed 97.5 percent of aphids by day five, the highest mortality at that time point among all isolates, even though it started more slowly, causing 20 percent mortality at day three compared with just 1.25 percent for MaCMUIPL 43. Based on this rapid action, the researchers selected MaCMUIPL 57 for a full virulence test across a range of spore concentrations from 1 × 10⁵ to 1 × 10⁹ conidia per milliliter. The response was clearly dose-dependent. At the highest concentration, mortality reached 100 percent within five days, and at 1 × 10⁸ conidia per milliliter it reached 97.5 percent after 120 hours. Lower concentrations of 1 × 10⁷, 1 × 10⁶, and 1 × 10⁵ conidia per milliliter produced 97.5, 87.5, and 80 percent mortality respectively at five days, and every concentration eventually achieved complete mortality by day seven.

Sporulation capacity, a key trait for mass production and field persistence, also favored MaCMUIPL 57. The three most virulent isolates were grown on potato dextrose agar and assessed at 7, 10, and 14 days of incubation. At seven days, MaCMUIPL 57 led with 10.5 × 10⁶ conidia per milliliter, ahead of MaCMUIPL 14 at 8.5 × 10⁶ and MaCMUIPL 37 at 3.7 × 10⁶. MaCMUIPL 14 briefly took the lead at day ten with 22.6 × 10⁶ conidia per milliliter, but by day fourteen MaCMUIPL 57 had produced the numerically highest yield of the experiment, 52.2 × 10⁶ conidia per milliliter, compared with 44.2 × 10⁶ for MaCMUIPL 14 and 20.5 × 10⁶ for MaCMUIPL 37. The authors note that total colony production and colony-level conidial yield were not assessed, which limits interpretation of overall sporulation potential.

Heat tolerance proved to be the isolate’s most distinctive trait. Conidial suspensions were exposed to 45 degrees Celsius in a water bath for up to 120 minutes, after which germination was measured on agar plates. All isolates began with 100 percent germination, but their fates diverged sharply under thermal stress. After 30 minutes, MaCMUIPL 57 retained 76.5 percent germination, far ahead of MaCMUIPL 14 at 31.75 percent and MaCMUIPL 37 at 23 percent. By 60 minutes, germination had fallen to 25.75 percent for MaCMUIPL 57 while the other two isolates dropped below 7 percent. At 90 minutes, only MaCMUIPL 57 and MaCMUIPL 37 showed any germination, at 12.25 and 2 percent respectively, and after 120 minutes MaCMUIPL 57 was the sole isolate with residual germination, at 2 percent. Because these fungi generally grow best at 25 to 30 degrees Celsius and require high humidity, the authors argue that such resilience matters for deployment in tropical and subtropical agroecosystems where thermal stress frequently undermines fungal persistence.

Molecular characterization added a genetic dimension to the phenotypic results. The team sequenced the GAS1 gene, a virulence-associated gene encoding a glycosylphosphatidylinositol-anchored β-1,3-glucanosyltransferase involved in building and reshaping the fungal cell wall. The GAS1 fragments from MaCMUIPL 14, 37, and 57 showed 100 percent sequence similarity to the GAS1 gene of Beauveria bassiana reference sequences in GenBank, and 98.83 to 98.92 percent similarity to another B. bassiana reference, confirming the gene’s presence and conservation across these entomopathogenic fungi. The primers used were originally designed for Beauveria species, and the authors note that understanding of this gene in Metarhizium remains limited, though cross-amplification across related hypocrealean fungi is feasible.

Gene expression analysis pointed toward mechanisms behind the isolate’s stress tolerance and productivity. Using reverse transcription quantitative PCR with the 18S rRNA gene as an internal control, the researchers profiled cag8, a conidiation-associated gene linked to thermotolerance, and slt2, a serine/threonine protein kinase gene involved in cell wall integrity and stress adaptation. MaCMUIPL 57 showed the highest expression of both genes among the tested isolates. Pearson correlation analysis revealed a strong positive correlation between cag8 expression and conidial germination after 30 minutes of heat exposure, with r = 0.99999 and p = 0.0028 across three isolates, though the authors caution that the small sample size warrants careful interpretation. The association between slt2 expression and conidial production at 14 days was positive but not statistically significant, with r = 0.750 and p = 0.460, and the authors state that larger studies are needed to confirm it.

The authors are careful to frame these results within their limits. All findings come from controlled laboratory assays at 25 ± 2 degrees Celsius, and the study did not test formulated products, shelf life, or field performance. They acknowledge that exceptional laboratory performance, whether rapid mortality, high sporulation, or elevated stress tolerance, does not always translate directly into consistent field efficacy, because ecological trade-offs and complex biotic and abiotic interactions in agroecosystems can constrain fungal persistence. They also note that competing hypotheses remain about whether isolate performance is governed primarily by specific regulatory pathways or by broader multigenic metabolic networks. Mortality data were corrected using Abbott’s formula, analyzed with repeated-measures general linear models, and compared with Tukey’s HSD tests, with conidial yield and thermotolerance assessed by two-way ANOVA.

Even with those caveats, the study offers a concrete template for identifying elite fungal biocontrol candidates. Rather than selecting isolates on pathogenicity alone, the team combined virulence against the target pest with conidial yield, thermotolerance, and expression analysis of key virulence- and stress-associated genes, an approach they argue gives a more comprehensive picture of candidate quality. MaCMUIPL 57, with its rapid kill at high doses, complete mortality from even the lowest tested concentration within a week, leading sporulation at day fourteen, and the only surviving germination after two hours at 45 degrees Celsius, stands out as the strongest performer in this screen. The authors conclude that future research should prioritize formulation development, stability and shelf-life assessment, field efficacy under diverse environmental conditions, and compatibility with integrated pest management strategies before the isolate can move toward commercial application against the mustard aphid.

Subject of Research: Screening native Metarhizium anisopliae isolates for biological control of the mustard aphid Lipaphis erysimi

Article Title: Integration of pathogenicity, thermotolerance, and gene expression analyses reveals a superior native Metarhizium anisopliae isolate for the biological control of the mustard aphid, Lipaphis erysimi

Article References: Pittarate, S., Siripan, T., Vivekanandhan, P., Swathy, K., Suwannarach, N., Kumla, J., Priyashantha, A. K. H., Lin, C.-H., Lin, H.-Y., Chang, F.-M., Nai, Y.-S., & Krutmuang, P. (2026). Integration of pathogenicity, thermotolerance, and gene expression analyses reveals a superior native Metarhizium anisopliae isolate for the biological control of the mustard aphid, Lipaphis erysimi. Frontiers in Fungal Biology, 7, Article 1929653. https://doi.org/10.3389/ffunb.2026.1929653

Image Credits: AI Generated

DOI: 10.3389/ffunb.2026.1929653

Keywords: Metarhizium anisopliae, mustard aphid, Lipaphis erysimi, biological control, entomopathogenic fungi, thermotolerance, conidial yield, GAS1 gene, cag8, slt2, virulence, integrated pest management