Mycelial compatibility, anastomosis, and nucleus numbers of eight Mexican Hirsutella citriformis strains isolated from Diaphorina citri

Abstract

Background: Among entomopathogenic fungi, H. citriformis has been recognized as potential biocontrol agent against the Asian citrus psyllid Diaphorina citri (Hemiptera: Liviidae). Nevertheless, this fungus is poorly characterized. Previous molecular studies have shown high sequence similarities among strains, but significant differences in Diaphorina citri virulence.

Objective: The aim of the present study was to determine mycelial compatibility and anastomosis, and nucleus numbers in mycelium and conidia of eight H. citriformis strains isolated from mycosed D. citri adults collected from several Mexican states.

Methods: Mycelial compatibility and anastomosis evaluation was performed after pairing strains, leading to 36 confrontations, and cultured in chlorate minimum medium to obtain mutants for vegetative compatibility group.

Results: Hypha or conidia nuclei were visualized with safranin-O and 3% KOH, and 0.05% trypan blue-lactophenol solution. H. citriformis strains showed compatibly and anastomosis events after confrontation. In addition, they showed one nucleus per conidium and mycelium section. It was not possible to obtain H. citriformis nit mutants from the chlorate concentrations tested.

Conclusions: To date, this is the first report demonstrating mycelial compatibility, anastomosis occurrence, and hyphae and conidia nuclei number among H. citriformis strains.

Keywords: Fungus mexican native strains; Mycelial characterization; Nit mutants; Nuclei.

Figure 1. Mycelial compatibility among Hirsutella citriformis strains.

(A, C & D) Mycelial compatibility between two colonies from one strain (INIFAP-Hir 5 vs INIFAP-Hir-5, IB-Hir-2 vs IB-Hir-2, or INIFAP-Hir 7 vs INIFAP-Hir-7, respectively). (B) Mycelial compatibility between the IB-Hir-2 vs INIFAP-Hir-5 strains. (E) Mycelial compatibility between the IB-Hir-2 vs INIFAP-Hir-7 strains. (F) Mycelial compatibility among the eight H. citriformis tested strains.

Figure 2. Different interactions observed by the Hirsutella citriformis strain INIFAP-Hir-1 anastomosis.

(A) Anastomosis event occurred between hyphal tip with lateral portion of other hyphae; (B) anastomosis parallel bridges between hyphae.

Figure 3. Hirsutella citriformis strain INIFAP-Hir-4 hyphal growth in restrictive media.

(A) Wild-type strain growth in minimum medium (MM); (B) colony growth in chlorate minimum medium (MM-C).

Figure 4. Phenotypic characterization of Hirsutella citriformis strain INIFAP-Hir-2 on selective media for nit mutants.

Strain was exposed to chlorate minimum medium (MM-C) at 3% potassium chlorate, and transferred to minimal medium (MM) supplemented with the following nitrogen sources to characterize nit mutants, (A) 2 g/L nitrate = MM-N, (B) 0.5 g/L nitrite = MM-NI, (C) 1.6 g/L ammonium tartrate = MM-TA, (D) 0.2 g/L hypoxanthine = MM-H, and (E) 0.2 g uric acid = MM-UA. It was also exposed to MM-C at 2.5% potassium chlorate, and transferred to MM with the same nitrogen sources as described above, where (F) 2 g/L nitrate = MM-N, (G) 0.5 g/L nitrite = MM-NI, (H) 1.6 g/L ammonium tartrate = MM-TA, (I) 0.2 g/L hypoxanthine = MM-H, and (J) 0.2 g uric acid = MM-UA, or exposed to MM-C without potassium chlorate, and transferred to MM with the same nitrogen sources as described above, where (K) 2 g/L nitrate = MM-N, (L) 0.5 g/L nitrite = MM-NI, (M) 1.6 g/L ammonium tartrate = MM-TA, (N) 0.2 g/L hypoxanthine = MM-H, and (O) 0.2 g uric acid = MM-UA.

Figure 5. Hirsutella citriformis strain IB-Hir-2 hyphal growth.

Strain was stained with (A) safranin O and potassium hydroxide, where arrows indicate nuclei; (B) lactophenol trypan blue (1.2 mL of liquified phenol, 10 mL of lactic acid, 8.8 mL of distilled water, and 0.02 g trypan blue), where arrows indicate mycelium septums; (C) IB-Hir-2 hyphal stained with safranin O and potassium hydroxide, where arrows indicate the nucleus inside a conidium.