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

Figures & data

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.

Table 1. LC₅₀ values (conidia/mL) for A. nomius against different life-cycle stages of O. furnacalis..

Insect stage	χ^2 †	LC50 ‡	LC50 confidence Interval		Concentration response (conidia/mL)
					Test for slope			Test for intercept
			Lower	Upper	Slope ± SE	P	95% CI	Intercept ± SE	P	95% CI
1^st instar	1.217	1.42 × 10^5 b	1.37 × 10^3	4.89 × 10^6	0.67 ± 0.25	0.007	0.176–1.158	−3.44 ± 1.69	0.042	−5.125 to − 1.749
2^nd instar	0.495	2.17 × 10^5 b	1.03 × 10^4	8.80 × 10^6	0.97 ± 0.36	0.006	0.267–1.676	−5.19 ± 2.22	0.019	−7.403 to − 2.971
3^rd instar	4.080	8.52 × 10^3 a	6.03 × 10^2	9.12 × 10^4	0.24 ± 0.01	0.349	−0.262 to 0.741	−0.94 ± 2.70	0.727	−3.639 to 1.757
4^th instar	0.064	2.30 × 10^8 d	1.68 × 10^7	5.79 × 10^11	0.45 ± 0.21	0.034	0.032–0.861	−3.73 ± 1.68	0.026	−5.413 to − 2.056
5^th instar	0.104	2.19 × 10^8 d	1.21 × 10^7	3.79 × 10^12	0.24 ± 0.15	0.110	−0.055 to 0.545	−2.04 ± 1.37	0.136	−3.412 to − 0.672
Pupae	0.021	8.20 × 10^7 c	5.34 × 10^5	8.97 × 10^11	0.11 ± 0.01	0.462	−0.188 to 0.414	−0.89 ± 2.22	0.687	−3.109 to 1.322
Adult	2.456	2.00 × 10^5 b	1.54 × 10^3	3.97 × 10^6	0.13 ± 0.01	0.387	−0.166 to 0.428	−0.66 ± 2.19	0.764	−2.841 to 1.530

†Chi-squared; ‡LC₅₀ of one treatment was significantly different if the lower and upper fiducial limits do not include the LC₅₀ value of other treatments; the LC₅₀ values are expressed as the concentration of conidia mL⁻¹. Data on LC₅₀ within a column followed by the same small letters were not significantly different (factorial ANOVA; Tukey’s test at p ≤0.05). LC₅₀, median lethal concentration.

Table 2. LT₅₀ values (days) of A. nomius against different life-cycle stages of O. furnacalis..

Insect stage	Chi-squared (χ^2)	Concentration response (conidia/mL)
		LT50	LT50 95% CI		Test for slope			Test for intercept
			Lower	Upper	Slope ± SE	p	95% CI	Intercept ± SE	p	95% CI
1^st instar	4.554	3.90 a	2.80	4.95	3.33 ± 0.73	0.000	1.894–4.775	−1.97 ± 0.53	0.000	−2.495 to − 1.443
2^nd instar	1.323	4.42 ab	3.53	5.33	5.01 ± 1.13	0.000	2.792–7.219	−3.23 ± 0.79	0.000	−4.026 to − 2.438
3^rd instar	2.264	4.91 b	4.04	5.80	4.58 ± 0.85	0.000	2.911–6.253	−3.17 ± 0.65	0.000	−3.814 to − 2.521
4^th instar	0.559	7.23 d	5.54	9.37	3.58 ± 1.01	0.000	1.603–5.561	−3.076 ± 0.89	0.001	−-3.964 to − 2.189
5^th instar	2.093	7.16 d	5.75	8.83	3.08 ± 0.57	0.000	1.959–4.196	−2.630 ± 0.513	0.000	−3.143 to − 2.188
Pupae	0.091	8.88 e	6.88	14.84	2.88 ± 0.83	0.001	1.257–4.499	−2.730 ± 0.704	0.000	−3.434 to − 2.026
Adult	1.605	6.35 c	5.06	8.04	3.03 ± 0.60	0.000	1.862–4.193	−2.43 ± 0.492	0.000	−2.922 to − 1.938

LT₅₀, median lethal time; CI, confidence interval.

Data on LT₅₀ within a column followed by the same small letters were not significantly different (factorial ANOVA; Tukey’s test at p ≤ 0.05).

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.

Data Availability statement

The data used to support the findings of this study are available from the corresponding author upon request.