Fungus Shows Laboratory Virulence Against Citrus Psyllid Vector of Huanglongbing

The Asian citrus psyllid, Diaphorina citri, is one of the most consequential insect pests in global citrus production, not primarily because of the direct damage it inflicts on trees, but because it serves as the primary vector of Candidatus Liberibacter asiaticus, the phloem-limited bacterium responsible for Huanglongbing, also known as citrus greening. The disease has caused severe economic losses across citrus-producing regions of Asia, Africa, and the Americas. Managing the psyllid has long depended on intensive chemical insecticide programs, with foliar applications typically made six to eight times per year against adults and nymphs, using organophosphates, pyrethroids, and systemic neonicotinoids such as imidacloprid. These measures struggle to keep pace with a pest that multiplies rapidly and readily develops insecticide resistance, which is why researchers continue to search for biological alternatives that can be folded into integrated pest management programs.

A new laboratory study published in the Egyptian Journal of Biological Pest Control examines one such alternative: the entomopathogenic fungus Akanthomyces lecanii, formerly known as Verticillium lecanii and Lecanicillium lecanii. Researchers at Fujian Agriculture and Forestry University in Fuzhou, China, together with collaborators, tested two isolates of the fungus, designated V3450 and V09, against eggs, nymphs, and adults of D. citri under controlled conditions. Their aim was twofold: to compare the virulence of the two isolates across the psyllid’s developmental stages, and to characterize, using light and scanning electron microscopy, how the more virulent isolate attaches to and develops on the insect’s body. The work positions A. lecanii as a candidate microbial control agent, while the authors are careful to frame their findings as a laboratory basis for further greenhouse and field evaluation rather than proof of practical field efficacy.

The experimental system was built on a psyllid colony originally collected from citrus trees in Fuzhou and maintained for four successive generations on orange jasmine, Murraya paniculata, at 25 degrees Celsius, 60 percent relative humidity, and a 14-hour light, 10-hour dark photoperiod. The two fungal isolates came from the laboratory’s own culture collection and were identified through a combination of morphological examination and molecular analysis of the internal transcribed spacer region: single discrete amplicons of 588 and 574 base pairs were obtained for V3450 and V09 respectively, and the sequences have been deposited in GenBank under accession numbers EU284716.1 and EU284718.1. Conidial suspensions were prepared from cultures grown on Czapek Dox agar and adjusted with a hemocytometer to five concentrations ranging from 1 × 10⁴ to 1 × 10⁸ conidia per milliliter. Only suspensions showing at least 95 percent germination on potato dextrose agar were used in the bioassays.

Inoculation was carried out by immersing eggs, nymphs, or adults in the conidial suspension, or in a 0.01 percent Tween 80 control solution, for 30 seconds. Each treatment comprised four independent replicates of 20 individuals, giving 80 insects or eggs per treatment in a completely randomized design. Treated material was incubated at 25 ± 1 °C and 70 ± 2 percent relative humidity for seven days, and mortality was corrected for natural background mortality using Abbott’s formula; final control mortality ranged from 5 to 15 percent across developmental stages. Statistical comparisons relied on one-way ANOVA followed by Tukey’s honestly significant difference test, while median lethal concentrations and median lethal times were estimated by probit regression with 95 percent confidence intervals.

The central result was a clear concentration-dependent pattern: mortality rose with both increasing conidial concentration and longer exposure time, and isolate V3450 was consistently more pathogenic than V09 across all tested stages. The most striking response came from nymphs, the most susceptible stage. Both isolates achieved 100 percent corrected mortality of third- and fourth-instar nymphs at the highest concentration of 1 × 10⁸ conidia per milliliter, against 15 percent mortality in the control group. Corrected nymphal mortality ranged from 81 to 100 percent for V3450 and 80 to 100 percent for V09 over the seven-day observation period. At that same top concentration, the median lethal times were 2.74 days for V3450 and 2.33 days for V09. Notably, because mortality was already high even at the lowest tested concentration, the researchers could not estimate reliable LC₅₀ values for nymphs within the concentration range they used, and they caution that the extrapolated nymphal LC₅₀ values should not be treated as precise potency estimates.

Adult psyllids were less susceptible than nymphs but still responded strongly at high doses. The LC₅₀ against adults at seven days post-treatment was 1.89 × 10⁴ conidia per milliliter for V3450 and 5.43 × 10⁴ conidia per milliliter for V09, and the median lethal times at the highest concentration were 4.28 and 3.36 days respectively. Corrected adult mortality over seven days spanned 36.67 to 96.25 percent for V3450 and 18.33 to 95 percent for V09 across the concentration series. Microscopic observations showed that conidia preferentially accumulated on the ventral surface of adults, particularly around the bases of the mouthparts and the leg joints, leading the authors to suggest that these regions, with their thinner cuticular structures, may serve as preferential entry points for fungal development. By 168 hours after inoculation, most infected adults were covered in mycelium, and in some cases the wings had separated from the body.

Eggs proved the most resistant stage. After seven days, the highest concentration produced an egg-hatching inhibition rate of 52 percent for V3450 and 50 percent for V09, with significant differences among the five concentrations for both isolates. The corresponding LC₅₀ values were 8.48 × 10⁷ conidia per milliliter for V3450 and 1.78 × 10⁸ for V09, roughly three to four orders of magnitude higher than the adult LC₅₀ values. Infected eggs shrank and turned orange-brown, with fungal outgrowths visible on the surface of most treated eggs. The authors emphasize that the mechanism behind the hatching inhibition remains unresolved: it could reflect direct fungal effects on the eggs, activity of fungal metabolites, or a combination of both, and they call for metabolite analysis and chorion structural assessment to distinguish among these possibilities. They also note that the relatively low ovicidal effect may be associated with the protective properties of the egg chorion, which can limit fungal establishment.

The microscopy component, conducted only with the more virulent V3450, documented the infection sequence chronologically at 24, 48, 72, 96, 120, 144, and 168 hours post-inoculation. At 24 hours, conidial attachment and germ-tube emergence were visible; germ tubes elongated across the cuticular surface by 48 hours; external hyphal development became evident at 72 hours; and by 96 hours, hyphae were associated with cuticular cracks and localized swellings that morphologically resembled appressoria, although the authors stress that the identity of these structures requires confirmation with higher-resolution techniques. External mycelial growth increased through 120 and 144 hours, and by 168 hours dense fungal colonization covered the body surface, with conidia produced on the insect and the body drying out. Fungal structures were observed on the wings, ovipositor, compound eyes, cuticle, and genital region. Light microscopy confirmed branched hyphae and conidia on the wing surface and along the wing veins, with hyphal morphology on the wings resembling that seen on culture medium.

The authors are explicit about the limits of what these observations establish. The SEM and light microscopy work was qualitative and documented representative patterns of adhesion, germination, and hyphal development, but did not quantify germination frequency, penetration events, or temporal variation. Uninfected controls processed under identical microscopy conditions were not included, and the surface observations do not demonstrate internal fungal colonization; histological sections, fluorescence-based localization, or transmission electron microscopy would be needed to confirm that the fungus invades internal tissues. The physiological basis of V3450’s greater virulence also remains unknown, since germination kinetics, enzyme activity, and secondary metabolite profiles were not compared between the isolates. Non-target effects were likewise not evaluated, so safety to Tamarixia radiata and other natural enemies must be tested before any field application.

Within those constraints, the study offers a concrete laboratory benchmark: V3450 at 1 × 10⁸ conidia per milliliter is identified as a candidate for foliar-spray trials targeting D. citri nymphs, with greenhouse and field validation required before any role in Huanglongbing vector management. The authors point out that the immersion method used here provides uniform exposure but does not represent field spraying, that laboratory conditions of constant temperature and humidity do not reflect field fluctuations in temperature, humidity, and solar exposure that affect conidial viability, and that commercial development would require scalable conidial production, formulation stability, shelf life, field efficacy data, non-target safety, and regulatory compliance. As it stands, the work adds A. lecanii to the expanding repertoire of fungal biocontrol candidates for the citrus psyllid and provides a documented infection sequence against which future formulation and field studies can be measured.

Subject of Research: Laboratory evaluation of the entomopathogenic fungus Akanthomyces lecanii as a biological control agent against the Asian citrus psyllid Diaphorina citri

Article Title: Exploring the pathogenicity and infection processes of Akanthomyces lecanii against Diaphorina citri for biocontrol applications

Article References: Feng, Q., Ghafar, M. A., Wang, F., Hussain, M., Akutse, K. S., Wang, L., & Ismail, A. M. (2026). Exploring the pathogenicity and infection processes of Akanthomyces lecanii against Diaphorina citri for biocontrol applications. Egyptian Journal of Biological Pest Control, 36(1), Article 30. https://doi.org/10.1186/s41938-026-00911-5

Image Credits: AI Generated

DOI: 10.1186/s41938-026-00911-5

Keywords: Akanthomyces lecanii, Diaphorina citri, Asian citrus psyllid, Huanglongbing, entomopathogenic fungi, biological control, citrus pests, LC50, scanning electron microscopy, insect pathogenicity, integrated pest management, biopesticide