Analysis of the virulence, infection process, and extracellular enzyme activities of Aspergillus nomius against the Asian corn borer, Ostrinia furnacalis guenée (Lepidoptera: Crambidae)

Abstract

The control of Ostrinia furnacalis, a major pest of maize in Xinjiang, is challenging owing to the occurrence of resistant individuals. Entomopathogenic fungi (EPF) are natural insect regulators used as substitutes for synthetic chemical insecticides. The fungus Aspergillus nomius is highly pathogenic to O. furnacalis; however, its virulence characteristics have not been identified. This study aimed to analyse the lethal efficacy, mode of infection on the cuticle, and extracellular enzyme activity of A. nomius against O. furnacalis. We found that the mortality and mycosis of O. furnacalis were dose-dependent when exposed to A. nomius and varied at different life stages. The egg-hatching and adult emergence rates decreased with an increase in conidial suspension. The highest mortality (83.33%, 7 d post-infection [DPI]) and mycosis (74.33%, 7 DPI) and the lowest mortality response (8.52 × 10³ conidia mL^-1) and median lethal time (4.91 d) occurred in the 3^rd instar larvae of O. furnacalis. Scanning electron microscopy indicated that numerous conidia germination and infection structure formation may have contributed to the high pathogenicity of A. nomius against O. furnacalis. There were significant correlations between O. furnacalis mortality and the activities of extracellular protease, lipase, and chitinase of A. nomius. This study revealed the infection process of the highly pathogenic A. nomius against O. furnacalis, providing a theoretical basis and reference for strain improvement and field application of EPF.

Keywords: Aspergillus nomius; cuticle; extracellular enzymes activity; infection processes; lethal efficacy; ostrinia furnacalis.

Graphical abstract

Figure 1.

Effect of O. furnacalis eggs on the hatching rate and the survival, mortality, and mycosis of the 1^st instar larvae of O. furnacalis (n = 90) following spray infection with different A. nomius concentrations. (a) hatching rate of O. furnacalis eggs at 7 d post-infection (DPI) with A. nomius; (b and c) healthy eggs and mycosis of eggs; (d) survival probability of the 1^st instar larvae of O. furnacalis; (e) mortality and mycosis of the 1^st instar larvae of O. furnacalis infected withA. nomius; (f) A. nomiusmycosis of the 1^st instar larvae of O. furnacalis; (g and h) log-probit regression line of concentration-mortality and mycosis of the 1^st instar larvae of O. furnacalisto A. nomius.

Figure 2.

Survival, mortality, and mycosis of the 2nd and 3rd instar larvae of O. furnacalis (n = 90) following spray infection with different concentrations of A. nomius. (a and e) survival probability of the (a) 2nd and (e) 3rd instar larvae of O. furnacalis; (b and g) mortality and mycosis of the (b) 2nd and (g) 3rd instar larvae of O. furnacalis infected with A. nomius; (c and h) A. nomius mycosis on the (c) 2nd and (h) 3rd instar larvae of O. furnacalis; (d, e, i, and j) log-probit regression line of (d and i) concentration-mortality and (e and j) mycosis response of the 2nd and 3rd instar larvae of O. furnacalis to A. nomius.

Figure 3.

Survival, mortality, and mycosis of the 4^th and 5^th instar larvae of O. furnacalis (n = 90) following spray infection with different A. nomius concentrations. (a and f) survival probability of the (a) 4^th and (f) 5^th instar larvae of O. furnacalis; (b and g) mortality and mycosis of the (b) 4^th and (g) 5^th instar larvae of O. furnacalis infected with A. nomius; (c and h) A. nomius mycosis of the (c) 4^th and (h) 5^th instar larvae of O. furnacalis; (d, e, i, and j) log-probit regression line of (d and i) concentration-mortality and (e and j) mycosis response of 4^th and 5^th instar larvae of O. furnacalis to A. nomius.

Figure 4.

Survival, mortality, and mycosis of pupae and adults of O. furnacalis (n = 90) following spray infection with different A. nomius concentrations. (a and f) survival probability of O. furnacalis (a) pupae and (f) adults; (b and g) mortality and mycosis of O. furnacalis (b) pupae and (g) adults infected with A. nomius; (c and h) mycosis of A. nomius on O. furnacalis (c) pupae and (h) adults; (d, e, i, and j) log-probit regression line of (d and i) concentration-mortality and (e and j) mycosis response of O. furnacalispupae and adult to A. nomius.

Figure 5.

Effect of infection with A. nomius on the rate of pupa eclosion and pupa development of O. furnacalis.(a) pupa eclosion rate of O. furnacalis and malformed adults ofO. furnacalis emerged in a group of pupae infected with different A. nomius concentrations; (b) malformed O. furnacalis adults; (c) healthy O. furnacalis adults.

Figure 6.

Cuticle topography of the 3^rd instar larvaeofO. furnacalis. (a) full view of a larva showing strumae and gentle surface topography; (b) gentle surface topography; (c) strumae surface topography; (d and e) sites beside the setae and proleg; (f) head capsule.

Figure 7.

Conidial attachment and germination of A. nomius on the cuticle of the 3^rd instar larvae of O. furnacalis observed under a scanning electron microscope. (a) conidium (Co) attached to the setal alveolus and the germination of a few conidia with formed germ tube (gt) within 8 h and appressorium (ap) at 24 h post-inoculation; seta = se. (b) appressorium (ap) and penetration pegs (pp) formed at 36 h post-inoculation. (c and d) numerous conidia germinated to form a germ tube (gt), conidial germination rates increased, and mycelium formed on the host surface within 36 h; penetration pegs = pp, hyphae = hy.

Figure 8.

Penetration and conidia development and secondary conidiogenous structure of A. nomiuson the cuticle surface of the 3^rd instar larvae ofO. furnacalis. (a) several hyphae penetrating the body wall from the setal alveolus outward from the body at 60 h; hyphae = hy. (b) hyphae of A. nomiusdeveloped, branched, and formed a dense mycelial mass on the cuticle 72 h after inoculation; hyphae = hy. (c and d) secondary conidiogenous structure formation of A. nomius84 h after inoculation; phialides = Ph, conidiophores = cp, vesicle = ve.

Figure 9.

Scanning electron representative microscopy images of the 3^rd instar larvae of O. furnacalis infected with A. nomius(120 h post-infection). (a) head capsule with abundant conidia and mycelial growth. (b) Extensive growth of mycelia in the abdomen. (c and d) full view of a larva showing complete mycelial networking.

Figure 10.

Activities of extracellular enzymes and their correlation with the virulence of A. nomius. (a) lipase activity, (b) protease activity, (c) chitinase activity, (d) relationship between the mortality rate of the 3^rd instar larvae of O. furnacalis and lipase activity, (e) relationship between the mortality rate of the 3^rd instar larvae of O. furnacalis and protease activity, (f) relationship between the mortality rate of 3^rd instar larvae of O. furnacalis and chitinase activity.