The pathogen and pathogenicity
Field observations conducted in pome orchards indicated the presence of infected and dead larvae of the leopard moth, Z. pyrina, which exhibited abnormalities in color, size, and shape. Dead larvae were rigidly mummified. Some of the dead larvae were found sticking at/near the openings of galleries on branches and covered with white thin mycelial growth of Beauveria. Larvae of Z. pyrina, collected from pome orchards, were found to be infected with the EPF, B. bassiana, which is commonly referred to as the white muscardine fungus. Alive infected larvae were feeble with large irregular dark brown areas on the cuticle. In Indonesia, Utomo et al. (1988) isolated a strain of Beauveria sp. from the dead larvae of the cocoa red borer, Zeuzera coffeae. Zhu and Ma (1985) isolated B. bassiana from dead larvae of Carpocapsa pomonella collected from apple orchards. The isolated strain was highly pathogenic to different lepidopterous larvae including Z. pyrina. In a related study, Sewify and Sharaf El-Din (1993) reported that larvae of Z. pyrina were susceptible to the infection with Metarhizium anisopliae under laboratory conditions.
Prevalence of Beauveria in larvae of Zeuzera
Although, the prevalence of Beauveria in larvae of Zeuzera was higher in the second year than in the first one, there was insignificant difference between the 2 years of survey, except mortalities of November and January in the mixed orchard (P ≤ 0.05). Obtained data of the present study revealed that in two successive years, there were significant differences in the prevalence of Beauveria in larval population of Zeuzera (P ≤ 0.05) between the two investigated farms (Fig. 1). Generally, it seems that larval mycoses are density-dependent. For example, first larval infection was recorded in the second week of September, but the fungus prevalence was low (1.9%) and increased gradually to reach the highest mortality (7.2%) in December (Fig. 2). In both years of study, the peak of fungus infection was recorded in December and January reaching 7.2 and 5.9%, respectively. It was coincident with the peak of population density of larvae. Although, the olive farm was ecologically managed, in both years of survey, no infections were recorded from May to September. In winter, the epizootics ranged from 1.4 to 3.1%, whereas it ranged between 0.4 and 1.3% in spring, and 0.3 and 2.1% in autumn. However, insignificant differences were recorded in the prevalence of Beauveria in larval population of Zeuzera (P ≤ 0.05) between the 2 years of survey in olive farm. Olive trees were approximately 10 years old. The low mortality percentage among Zeuzera larvae could be attributed to low load of Beauveria inoculum in the newly reclaimed desert lands. Epizootics recorded in this farm was comparable to that recorded (1.6%) by Hegazi et al. (2014).
Likely, the behavior of larvae, e.g., overcrowding in galleries, abundance of young susceptible larvae and the dominant environmental conditions, particularly during winter allowed a successful infection and easily spread of Beauveria within larval population. In this context, the field dissection of infested olive branches showed that a large number of leopard larvae of different instars remained in a dormant stage for overwintering (Hegazi et al. 2014). The same authors have investigated 245 Zeuzera larvae in September and recorded that 1.6% of them infected with B. bassiana. During the first year, no larval mortalities were recorded in the period from May to September, while 1.9% was recorded during September of the second year. This could be attributed to behavior of larvae and the dominant environmental conditions in summer months (Sarto 2001). Ecological studies revealed that most of Zeuzera larvae were much smaller, continuing to feed throughout the season and become fully grown only in the late summer (Ismail et al. 1992; Hegazi et al. 2009 and Merghem and Ahmed 2017). In Spring, the prevalence of Beauveria in larvae of Zeuzera was significantly less than in winter which may be due to the larvae that started feeding and boring into branches, and majority of larvae became full-grown and ready for pupation, thus may be more resistant to infection. The low activity of the naturally occurring Beauveria in spring and summer may be due to negative impacts of low host density and/or reduced fungal inoculum load in sprayed or sanitized pome orchards (Hegazi et al. 2014). The same trend was also reported by (Wraight et al. 2018).
The inoculums source may be the larval cadavers in galleries or fungus spores occurring in orchard’s soil. However, regarding the fungus transmission, likely by direct contact between active larvae and cadavers embedded in galleries. Sun et al. (2008) surveyed a total of 20 species of insect-associated fungi in orchard soils. The survey included insect-pathogenic fungi and opportunistic insect pathogens. They recorded three insect-pathogenic species, B. bassiana, Metarhizium anisopliae, and Paecilomyces fumosoroseus.
Histopathological analysis
The insect cuticle is a highly heterogeneous structure that can vary greatly in composition even during the various life-stages of a particular insect. The EPF induce infection via penetration essentially anywhere on the host cuticle, although preferential sites have been noted on various insects (Ashtari et al. 2011 and Sahayaraj et al. 2013). Histopathological studies indicated that the fungus conidia germinated on the cuticle surface with a thin germ hypha which penetrated into the cuticle 2 days after contamination (Fig. 3a). Ortiz-Urquiza and Keyhani (2013) reported that infection begins with attachment of single-celled dispersive forms of the fungus, e.g., conidia or blastospores, to the insect cuticle. However, the penetration was observed mainly on the intersegmental areas and at sites where masses of germ hyphae were detected, probably due to the absence of cuticulin in this area of the integument (Fig. 3b, c). The epicuticle layer provides a hydrophobic barrier rich in lipids and is followed by the procuticle that contains chitin and sclerotized protein (Hajek and Leger 1994 and Ortiz-Urquiza and Keyhani 2013). Elsayed et al. (1989) detected high levels of both endo- and exo-chitinase activity in penetration sites of Nomuraea rileyi into larva of cabbage looper, Trichoplusia ni. On the penetration sites, the outer part of the cuticle changed into a dark brown color (dark spots), while the endocuticle appeared degraded near the penetrant hyphae which indicates the formation of appressorium (Fig. 4c, d). In contrast, Schneider et al. (2013) reported that the adhesion stage and formation of the appressorium noticed 18 h after the infection, occasioning black spots, and depressions in the cuticle of young and old infected pupae of Diatraea saccharalis. Similarly, Vega et al. (2015) mentioned that B. bassiana was accomplished penetration into the coffee berry borer, Hypothenemus hampei, within a few hours, but required several days to infect and kill it. Six days after infection, the saprophytic phase of Beauveria was recorded. The hyphal bodies and blastospores of the fungus were observed in large numbers in the body cavity. Prior to death, the hyphae were abundant in most of the body tissues. After death, the larvae cadavers were soft and pliable but became rigid and mummified within 24 h at 25 °C. When rigid cadavers were maintained at 25 °C and 100% R.H., they were completely covered by the fungus mycelia within 4–7 days (Fig. 4c). The development of B. bassiana into the larvae of Z. pyrina was similar to that recorded for the fungus in coffee berry borer H. hampei (Wraight et al. 2018) and the red palm weevil, Rhynchophorus ferrugineus (Dembilio et al. 2010). Müller-Kögler (1965) divided the life cycle of EPF into two phases; the parasitic phase in which the fungus infects and kills its host; and the saprophytic phase in which the fungus grows on the surface of the cadavers, producing conidiophores and conidia (Fig. 4d). The above two phases are distinct in the present study for Zeuzera larvae. Smears of hemolymph and fat bodies of infected larvae indicated the presence of short branched hyphae with dense cytoplasm and blastospores. When rigid cadavers were subjected to humid conditions (about 100% R.H.), the fungus grew on the surface of the cuticle with a white, fine, and thin mycelial growth. Within 2–3 days, the hyphae began to form spores. Most of the infected larvae were in their third instar. The occurrence of hyphal bodies of different sizes and dense cytoplasm in the body cavity penetrating all body tissues. Figure 3d illustrates the formation of the conidiophores bearing conidia spores and indicating the extrusion. Sewify and Sharaf El-Din (1993) found that the blastospores of M. anisopliae were noticed in hemolymph of infected larvae 24 h after infection and developed to hyphal bodies 36 h after infection.